Antifungal prophylaxis for cornea

ABSTRACT

The technology described herein is directed to ionic liquids for using the treatment or prevention of fungal infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. §120 of co-pendingUSSN 17/318,048 filed May 12, 2021, which claims benefit under 35 U.S.C.§ 119(e) of U.S. Provisional Application No. 63/023,374 filed May 12,2020 the contents of which are incorporated herein by reference in theirentireties.

TECHNICAL FIELD

The technology described herein relates to ionic liquids, e.g., cholineand undecanoic acid for treatment or prevention of funal infections.

BACKGROUND

After eye surgery, particularly keratoprosthesis (KPro) implantation,subjects can remain extremely suspectible to eye infections for the restof their life. Daily prophylactic antibiotics are common to preventbacterial infections, but affordable and tolerable antifungal agents arelacking.

SUMMARY

It is demonstrated herein that the ionic liquid choline : undecanoicacid is surprisingly effective against ocular fungal infections,virtually non-toxic, and both affordable and practical for dailylife-long use. The ionic liquid choline : undecanoic acid providesantifungal activity superior to a panel of tested agents, includingcurrent clinical standards.

Accordingly, described herein is a composition comprising at least oneionic liquid comprising a quaternary ammonium cation and an undecanoicacid anion. In some embodiments of any of the aspects, the cation has amolar mass equal to or greater than choline. In some embodiments of anyof the aspects, quaternary ammonium has the structure of NR₄ ⁺ and atleast one R group comprises a hydroxy group. In some embodiments of anyof the aspects, quaternary ammonium has the structure of NR₄ ⁺ and onlyone R group comprises a hydroxy group. In some embodiments of any of theaspects, the cation is choline, C1, C6, or C7.

In some embodiments of any of the aspects, the ionic liquid is at aconcentration of at least 0.1%w/v. In some embodiments of any of theaspects, the ionic liquid is at a concentration of from 10 to 70%w/v. Insome embodiments of any of the aspects, the ionic liquid is at aconcentration of from 30 to 50%w/v. In some embodiments of any of theaspects, the ionic liquid is at a concentration of from 30 to 40%w/v. Insome embodiments of any of the aspects, the ionic liquid comprises aratio of cation to anion of from 2:1 to 1:10. In some embodiments of anyof the aspects, the ionic liquid comprises a ratio of cation to anion offrom 2:1 to 1:2. In some embodiments of any of the aspects, the ionicliquid comprises a ratio of cation to anion of from 1:1 to 1:4. In someembodiments of any of the aspects, the ionic liquid has a cation:anionratio of 1:1.

In some embodiments of any of the aspects, the composition does notcomprise an active agent other than the at least one ionic liquid. Insome embodiments of any of the aspects, the composition furthercomprises at least one additional active compound other than the atleast one ionic liquid. In some embodiments of any of the aspects, theat least one additional active compound is an additional antifungalagent. In some embodiments of any of the aspects, the one or moreadditional antifungal agents are selected from the group consisting of:amphotericin B; natamycin; voriconazole; povidone-iodine; hypochlorousacid; Chlorhexidine digluconate (CDG); vancomycin (VAN); chloramphenicol(CHL); polymyxin B (PMB); trimethoprim (TMP); benzalkonium chloride(BAK); and combinations thereof.

In some embodiments of any of the aspects, the composition is formulatedfor administration transdermally, to a mucus membrane, orally, ocularly,to the cornea, subcutaneously, intradermally, parenterally,intratumorally, or intravenously. In some embodiments of any of theaspects, the composition is formulated for ocular administration.

The compositions described herein can be used to treat or prevent fungalinfections. Accordingly, described herein is a method of treating orpreventing a fungal infection, comprising administering to a subject inneed thereof a composition described herein. In some embodiments of anyof the aspects, the administration is transdermal, to a mucus membrane,oral, subcutaneous, intradermal, parenteral, intratumoral, ocular, orintravenous. In some embodiments of any of the aspects, the fungalinfection is an ocular fungal infection and the composition isadministered to one or both eyes. In some embodiments of any of theaspects, the subject is one who has received a corneal surgery. In oneaspect of any of the embodiments, described herein is a methodcomprising: performing corneal surgery on one or both eyes of a subject;and then administering a composition as described herein to the one orboth eyes. In some embodiments of any of the aspects, the surgery iskeratoprosthesis (KPro) implantation, artificial cornea surgery, orcornea replacement surgery.

In some embodiments of any of the aspects, the fungal infection isinfectious keratitis and/or endopthalmitis. In some embodiments of anyof the aspects, the fungal infection is an infection of Candida, Candidaalbicans, Candida parapsilosis, Fusarium, or Aspergillus. In someembodiments of any of the aspects, the composition is administereddaily. In some embodiments of any of the aspects, the composition isprovided in or on a contact lens, a lower conjunctival fornix device, ora subconjunctival device.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C demonstrate fungicidal activity against C. albicans (ATCC24433). FIG. 1A) Full-strength Polytrim®: the antibiotic Polytrim® andits components, PMB, TMP, and BAK were tested at full strength.Fungicidal activity was observed in a contact time-dependent manner,except for TMP, which had no effect. Reduction in viable colonies (<log₁₀) was observed within 15 min, with 2 log₁₀ and 3 log₁₀ reductionsobserved within 60 min and 120 min, respectively. FIG. 1B) Tenth-dilutedPolytrim®: fungicidal activity was not observed within the testedcontact times for tenth dilutions of Polytrim® and its components. FIG.1C) Comparator antibiotics: fungicidal activity was not observed withinthe tested contact times for other commonly used antibiotics (MOX, GAT,CHL, and VAN) without BAK. Viable colonies were counted after 24 hrs ofaerobic incubation at 37° C. on Sabouraud dextrose agar. PT = Polytrim®,PMB = polymyxin B sulfate, BAK = benzalkonium chloride, TMP =trimethoprim, MOX=moxifloxacin, GAT=gatifloxacin, CHL=chloramphenicol,VAN=vancomycin. Full strength Polytrim® contains 10,000 u/mL PMB, 40µg/mL BAK, and 1 mg/mL TMP. Concentration of MOX=5 mg/mL, GAT=5 mg/mL,CHL=5 mg/mL, VAN=14 mg/mL. Y-axis displays mean number (±SD) of viablecolonies in log₁₀ units from two to three independent experimentsperformed in technical triplicate. (to be done: p < value versus controlat time point by one-way analysis of variance and Tukey multiplecomparison test.)

FIGS. 2A-2D demonstrate the fungicidal activity and cytotoxicity of topfour candidates. FIG. 2A) Trypan Blue staining: confluent monolayers ofHCLE cells were stained with Trypan Blue following 1 min of contact withan antimicrobial agent. Displayed are mean values (±SD) of the % stainedarea per image. The top four candidates (Polytrim®, BAK, PI, and a novelIL) all had a significantly lower amount of staining (p-value) comparedto the control treatment (TX-100), as quantified by universal colorthresholding in FIJI™ software (n=3 - 6 images per treatment groupwithout overlapping areas). 10X images were obtained under phasecontrast microscopy, and results display two to three independentexperiments performed in technical duplicate. Fungicidal activityagainst FIG. 2B) C. albicans (ATCC 24433): PI and the novel IL caused a≥3 log₁₀ reduction in viable colonies within 1 min of contact. CDG wasalso fungicidal, causing a >log10 reduction within 1 min, but requiring5 min to have the same effect as PI and the novel IL. FIG. 2C) F. solani(MYA-3636): Polytrim® and PI caused a ≥3 log₁₀ reduction in viableconidia within 1 min of contact. BAK and the novel IL required more timeto have the same effect, but still sufficed to cause a 2 log₁₀ reductionwithin 1 min. FIG. 2D) A. fumigatus (MYA-3626): PI and the novel ILcaused a 2 log₁₀ and log₁₀ reduction in viable conidia within 1 min,respectively. CDG and Polytrim® had a very moderate effect (<log₁₀reduction) within 1 min and 15 min of contact, respectively.TX-100=Triton X-100 (1%), PBS=phosphate buffered solution (1X),PT=Polytrim®, BAK=benzalkonium chloride (40 µg/mL), PI=povidone iodine(0.5%), #3-IL=ionic liquid (10 mM), CDG=chlorhexidine digluconate(0.05%).

FIG. 3 depicts ratios of fungicidal activity/cytotoxicity. Displayed arethe calculated indices for the top candidates. PI and the novel IL hadthe highest ratios for all three of the tested species. Polytrim® andBAK were similar to PI for F. solani, and had the lowest ratio for C.albicans. CDG had the lowest ratio for A. fumigatus. The ratio iscalculated as follows: fungicidal activity/cytotoxicity. Fungicidalactivity=# of log₁₀ reduction in viable fungal cells,cytotoxicity=fold-change in Trypan Blue staining relative to the PBScontrol group (or % stained area for the antimicrobial/PBS) after 1 mincontact. PBS=phosphate buffered solution (1X), PT=Polytrim®,BAK=benzalkonium chloride (40 µg/mL), PI=povidone iodine (0.5%),#3-IL=ionic liquid (10 mM), CDG=chlorhexidine digluconate (0.05%).

FIG. 4 depicts the chemical formula of the IL. A 1:1 choline (C₅H₁₄NO)and undecanoic acid (C₁₁H₂₂O₂) ionic liquid at 10 mM in 1X PBS. It isstable at room temperature for months and tolerant of light exposure.The liquid is non-viscous and transparent with a slightly yellow tint. Amild odor is present due to the undecanoate component. (pH 7.2±0.2) Bothingredients are generally recognized as safe (GRAS) by the FDA. Cholineis an essential nutrient found in food and various biological pathwaysinvolved with cell membrane support, DNA synthesis, neurotransmission,etc. Both choline and undecanoic acid are cost-effective ingredientsalready being manufactured in large-scale.

FIG. 5 depicts fungal adhesion. Kontur™ soft contact lenses andPolytrim®: C. albicans (ATCC 24433) was pre-treated for 90 min with lowconcentrations of Polytrim®, and incubated with soft contact lensesafter extensive washing to remove traces of the antibiotic. Compared tothe non-treated control group, there was a reduction (>50%) in viablecolonies recovered from the surface of the soft contact lenses for boththe ⅒ and 1/100 strength Polytrim®-treated Candida groups. Control=softcontact lenses combined with PBS-treated C. albicans. Mean valuesdisplay the % reduction in number of viable colonies that were adheringto the surface of the soft contact lenses following aerobic incubationat 37° C. The post-sonified solutions containing the soft contact lenseswere streaked on Sabouraud dextrose agar to evaluate growth.

FIGS. 6A-6B depict tissue invasion. Denuded pig corneas weregamma-irradiated and incubated with C. albicans (ATCC 24433) to evaluatefungal penetration. Images display cryosections of the FIG. 6A)non-treated and FIG. 6B) gamma-irradiated (25 kGy) tissue taken underepifluorescence microscopy (4X) following calcofluor white staining.Infiltration of the Candida into the inner stromal layers was observedas faint, vertical lines (marked with arrows) in FIG. 6A). This stainingpattern was absent in FIG. 6B). Corneas were aerobically incubated withthe Candida for 48 hrs at 37° C., and tissue thickness was maintainedbetween the test groups with osmotically balanced medium containing 5%dextran (500 kDa MW).

DETAILED DESCRIPTION

As described herein, the ionic liquid choline:undecanoic aciddemonstrates surprising antifungal activity, particularly in a cornealenvironment. It is contemplated herein that the performance of thisionic liquid is surprisingly superior than that of undecanoic aciditself because the ionlic liquid is amphiphilic (as opposed to thehydrophobicity of the undecanoic acid), which provides a) improvedability to remain in suspension and b) superior penetration inbiological tissues. Accordingly, in one aspect of any of theembodiments, described herein is a composition comprising comprising atleast one ionic liquid comprising a quaternary ammonium cation and anundecanoic acid anion.

The term “ionic liquids (ILs)” as used herein refers to organic salts ormixtures of organic salts which are in liquid state at room temperature.This class of solvents has been shown to be useful in a variety offields, including in industrial processing, catalysis, pharmaceuticals,and electrochemistry. The ionic liquids contain at least one anionic andat least one cationic component. Ionic liquids can comprise anadditional hydrogen bond donor (i.e. any molecule that can provide an-OH or an - NH group), examples include but are not limited to alcohols,fatty acids, and amines. The at least one anionic and at least onecationic component may be present in any molar ratio. Exemplary molarratios (cation:anion) include but are not limited to 1 : 1, 1:2, 2: 1, 1:3, 3: 1, 2:3, 3:2, and ranges between these ratios. For furtherdiscussion of ionic liquids, see, e.g., Hough, et ah, “The thirdevolution of ionic liquids: active pharmaceutical ingredients”, NewJournal of Chemistry, 31 : 1429 (2007) and Xu, et al., “Ionic Liquids:Ion Mobilities, Glass Temperatures, and Fragilities”, Journal ofPhysical Chemistry B, 107(25): 6170-6178 (2003); each of which isincorporated by reference herein in its entirety. In some embodiments ofany of the aspects, the ionic liquid or solvent exists as a liquid below100° C. In some embodiments of any of the aspects, the ionic liquid orsolvent exists as a liquid at room temperature.

The cation of an IL described herein can be a cation comprising aquaternary ammonium. A quarternary ammonion is a positively chargedpolyatomic ion of the structure NR₄ ⁺, each R independently being analkyl group or an aryl group.

In some embodiments of any of the aspects, the cation has a molar massequal to or greater than choline, e.g., a molar mass equal to or greaterthan 104.1708 g/mol. In some embodiments of any of the aspects, thecation has a molar mass greater than choline, e.g., a molar mass equalgreater than 104.1708 g/mol.

In some embodiments of any of the aspects, each R group of thequarternary ammoniun independently comprises an alkyl, alkane, alkene,or aryl. In some embodiments of any of the aspects, each R group of thequarternary ammonium independently comprises an alkyl, alkane, oralkene. In some embodiments of any of the aspects, each R group of thequarternary ammoniun independently comprises an alkane or alkene. Insome embodiments of any of the aspects, each R group of thequaternernary ammonium independently comprises a carbon chain of no morethan 10 carbon atoms in length, e.g., no more than 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, or 30 carbon atoms in length. In someembodiments of any of the aspects, each R group of the quaternernaryammonium independently comprises a carbon chain of no more than 12carbon atoms in length. In some embodiments of any of the aspects, eachR group of the quaternernary ammonium idependently comprises a carbonchain of no more than 15 carbon atoms in length. In some embodiments ofany of the aspects, each R group of the quaternernary ammoniumindependently comprises a carbon chain of no more than 20 carbon atomsin length.

In some embodiments of any of the aspects, each R group of thequaternernary ammonium independently comprises a carbon chain of no morethan 10 carbon atoms, e.g., no more than 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, or 30 carbon atoms. In some embodiments of any of theaspects, each R group of the quaternernary ammonium independentlycomprises a carbon chain of no more than 12 carbon atoms. In someembodiments of any of the aspects, each R group of the quaternernaryammonium independently comprises a carbon chain of no more than 15carbon atoms. In some embodiments of any of the aspects, each R group ofthe quaternernary ammonium independently comprises a carbon chain of nomore than 20 carbon atoms.

In some embodiments of any of the aspects, each R group of thequaternernary ammonium independently comprises an alkyl group of no morethan 10 carbon atoms, e.g., no more than 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, or 30 carbon atoms. In some embodiments of any of theaspects, each R group of the quaternernary ammonium independentlycomprises an alkyl group of no more than 12 carbon atoms. In someembodiments of any of the aspects, each R group of the quaternernaryammonium independently comprises an alkyl group of no more than 15carbon atoms. In some embodiments of any of the aspects, each R group ofthe quaternernary ammonium independently comprises an alkyl group of nomore than 20 carbon atoms.

In some embodiments of any of the aspects, each R group of thequaternernary ammonium independently comprises an alkane, alkene, aryl,heteroaryl, alkyl, or heteroalkyl. In some embodiments of any of theaspects, each R group of the quaternernary ammonium independentlycomprises an unsubstituted alkane, unsubstituted alkene, unsubstitutedaryl, unsubstituted heteroaryl, unsubstituted alkyl, or unsubstitutedheteroalkyl. In some embodiments of any of the aspects, each R group ofthe quaternernary ammonium independently an unsubstituted alkane. Insome embodiments of any of the aspects, each R group of thequaternernary ammonium independently an unsubstituted alkene. In someembodiments of any of the aspects, each R group of the quaternernaryammonium independently comprises one or more substituent groups.

In some embodiments of any of the aspects, at least one R group of thequaternary ammonium comprises a hydroxy group. In some embodiments ofany of the aspects, one R group of the quaternary ammonium comprises ahydroxy group. In some embodiments of any of the aspects, only one Rgroup of the quaternary ammonium comprises a hydroxy group.

Exemplary, non-limiting cations can include choline and any of thecations designated C1-C7 which are defined by structure below.

Further non-limiting examples of cations include the following:

-   1-(hydroxymethyl)-1-methylpyrrolidin-1-ium-   1-(2-hydroxyethyl)-1-methylpyrrolidin-1-ium-   1-ethyl-1-(3-hydroxypropyl)pyrrolidin-1-ium-   1-(3-hydroxypropyl)-1-methylpyrrolidin-1-ium-   1-(4-hydroxybutyl)-1-methylpyrrolidin-1-ium-   1-ethyl-1-(4-hydroxybutyl)pyrrolidin-1-ium-   1-(4-hydroxybutyl)-1-propylpyrrolidin-1-ium-   1-(5-hydroxypentyl)-1-propylpyrrolidin-1-ium-   1-ethyl-1-(5-hydroxypentyl)pyrrolidin-1-ium-   1-(5-hydroxypentyl)-1-methylpyrrolidin-1-ium-   1-(hydroxymethyl)-1-methylpiperidin-1-ium-   1-(2-hydroxyethyl)-1-methylpiperidin-1-ium-   1-ethyl-1-(2-hydroxyethyl)piperidin-1-ium-   1-ethyl-1-(3-hydroxypropyl)piperidin-1-ium-   1-(3-hydroxypropyl)-1-propylpiperidin-1-ium-   1-(3-hydroxypropyl)-1-methylpiperidin-1-ium-   1-(4-hydroxybutyl)-1-methylpiperidin-1-ium-   1-ethyl-1-(4-hydroxybutyl)piperidin-1-ium-   1-(4-hydroxybutyl)-1-propylpiperidin-1-ium-   1-butyl-1-(5-hydroxypentyl)piperidin-1-ium-   1-(5-hydroxypentyl)-1-propylpiperidin-1-ium-   1-ethyl-1-(5-hydroxypentyl)piperidin-1-ium-   1-(5-hydroxypentyl)-1-methylpiperidin-1-ium-   3-ethyl-1-methyl-1H-imidazol-3-ium-   1-methyl-3-propyl-1H-imidazol-3-ium-   3-butyl-1-methyl-1H-imidazol-3-ium-   1-methyl-3-pentyl-1H-imidazol-3-ium-   1,2-dimethyl-3-pentyl-1H-imidazol-3-ium-   3-butyl-1,2-dimethyl-1H-imidazol-3-ium-   1,2-dimethyl-3-propyl-1H-imidazol-3-ium-   3-(hydroxymethyl)-1,2-dimethyl-1H-imidazol-3-ium-   3-(2-hydroxyethyl)-1,2-dimethyl-1H-imidazol-3-ium-   3-(3-hydroxypropyl)-1,2-dimethyl-1H-imidazol-3-ium-   3-(4-hydroxybutyl)-1,2-dimethyl-1H-imidazol-3-ium-   3-(5-hydroxypentyl)-1,2-dimethyl-1H-imidazol-3-ium-   3-(5-hydroxypentyl)-1-methyl-1H-imidazol-3-ium-   3-(4-hydroxybutyl)-1-methyl-1H-imidazol-3-ium-   3-(3-hydroxypropyl)-1-methyl-1H-imidazol-3-ium-   3-(2-hydroxyethyl)-1-methyl-1H-imidazol-3-ium-   3-(hydroxymethyl)-1,2,4,5-tetramethyl-1H-imidazol-3-ium-   3-(2-hydroxyethyl)-1,2,4,5-tetramethyl-1H-imidazol-3-ium-   3-(3-hydroxypropyl)-1,2,4,5-tetramethyl-1H-imidazol-3-ium-   3-(4-hydroxybutyl)-1,2,4,5-tetramethyl-1H-imidazol-3-ium-   3-(5-hydroxypentyl)-1,2,4,5-tetramethyl-1H-imidazol-3-ium-   1-(5-hydroxypentyl)pyridin-1-ium-   1-(4-hydroxybutyl)pyridin-1-ium-   1-(3-hydroxypropyl)pyridin-1-ium-   1-(2-hydroxyethyl)pyridin-1-ium-   1-(hydroxymethyl)pyridin-1-ium-   1-hydroxypyridin-1-ium-   (hydroxymethyl)trimethylphosphonium-   triethyl(hydroxymethyl)phosphonium-   triethyl(2-hydroxyethyl)phosphonium-   (2-hydroxyethyl)tripropylphosphonium-   (3-hydroxypropyl)tripropylphosphonium-   tributyl(3-hydroxypropyl)phosphonium-   (3-hydroxypropyl)tripentylphosphonium-   (4-hydroxybutyl)tripentylphosphonium-   (5-hydroxypentyl)tripentylphosphonium

In some embodiments of any of the aspects, the cation is choline, C1,C6, and/or C7. In some embodiments of any of the aspects, the cation isC1, C6, and/or C7. In some embodiments of any of the aspects, the cationis choline.

The anion of the ionic liquid described herein is undecanoic acid, alsoreferred to in the art as undecylic acid, is a carboxylic acid withchemical formula CH₃(CH₂)₉COOH and the following structure:

Non-limiting, exemplary combinations of cation and anions are providedin Table 1 below.

TABLE 1 Choline C1 C2 C3 C4 C5 C6 C7 Undecanoic acid x x x x x x x x

In some embodiments of any of the aspects, the IL is at a concentrationof at least 0.01% w/v. In some embodiments of any of the aspects, the ILis at a concentration of at least 0.05% w/v. In some embodiments of anyof the aspects, the IL is at a concentration of at least 0.1% w/v. Insome embodiments of any of the aspects, the IL is at a concentration ofat least 0.2% w/v, at least 0.3% w/v, at least 0.4% w/v, at least 0.5%w/v, at least 1% w/v or greater. In some embodiments of any of theaspects, the IL is at a concentration of from about 0.01% w/v to about1% w/v. In some embodiments of any of the aspects, the IL is at aconcentration of from 0.01% w/v to 1% w/v. In some embodiments of any ofthe aspects, the IL is at a concentration of from about 0.05% w/v toabout 0.5% w/v. In some embodiments of any of the aspects, the IL is ata concentration of from 0.05% w/v to 0.5% w/v.

In some embodiments of any of the aspects, the IL is at a concentrationof at least 25% w/w. In some embodiments of any of the aspects, the ILis at a concentration of at least 25% w/w in water. In some embodimentsof any of the aspects, the IL is at a concentration of at least 25% w/win saline or a physiologically compatible buffer.

In some embodiments of any of the aspects, the IL is at a concentrationof from about 5% w/w to about 75% w/w. In some embodiments of any of theaspects, the IL is at a concentration of from 5% w/w to 75% w/w. In someembodiments of any of the aspects, the IL is at a concentration of fromabout 5% w/w to about 75% w/w in water, saline or a physiologicallycompatible buffer. In some embodiments of any of the aspects, the IL isat a concentration of from 5% w/w to 75% w/w in water, saline or aphysiologically compatible buffer.

In some embodiments of any of the aspects, the IL is at a concentrationof at least about 0.1 % w/w. In some embodiments of any of the aspects,the IL is at a concentration of at least 0.1 % w/w. In some embodimentsof any of the aspects, the IL is at a concentration of from about 10 %w/w to about 70 % w/w. In some embodiments of any of the aspects, the ILis at a concentration of from 10 % w/w to 70 % w/w. In some embodimentsof any of the aspects, the IL is at a concentration of from about 30 %w/w to about 50 % w/w. In some embodiments of any of the aspects, the ILis at a concentration of from 30 % w/w to 40 % w/w. In some embodimentsof any of the aspects, the IL is at a concentration of from about 30 %w/w to about 50 % w/w. In some embodiments of any of the aspects, the ILis at a concentration of from 30 % w/w to 40 % w/w.

In some embodiments of any of the aspects, the % w/w concentration ofthe IL is % w/w concentration in water, saline, or a physiologicallycompatible buffer.

In some embodiments of any of the aspects, the IL is 100% by w/w or w/v.

In some embodiments, the IL is an anhydrous salt, e.g., an ionic liquidnot diluted or dissolved in water. In some embodiments, the IL isprovided as an aqueous solution.

In some embodiments of any of the aspects, the IL is at a concentrationof at least 25% w/w and has a ratio of cation:anion of at least 1:3. Insome embodiments of any of the aspects, the IL is at a concentration ofat least 25% w/w in water and has a ratio of cation:anion of at least1:3. In some embodiments of any of the aspects, the IL is at aconcentration of at least 25% w/w and has a ratio of cation:anion of 1:3or 1:4. In some embodiments of any of the aspects, the IL is at aconcentration of at least 25% w/w in water and has a ratio ofcation:anion of 1:3 or 1:4. In some embodiments of any of the aspects,the IL is a gel, or a shear-thining Newtonian gel.

In some embodiments of any of the aspects, the IL has a ratio ofcation:anion of from about 10:1 to about 1:10. In some embodiments ofany of the aspects, the IL has a ratio of cation:anion of from 10:1 to1:10. In some embodiments of any of the aspects, the IL has a ratio ofcation:anion of from about 5:1 to about 1:5. In some embodiments of anyof the aspects, the IL has a ratio of cation:anion of from 5:1 to 1:5.In some embodiments of any of the aspects, the IL has a ratio ofcation:anion of from about 2:1 to about 1:4. In some embodiments of anyof the aspects, the IL has a ratio of cation:anion of from 2:1 to 1:4.In some embodiments of any of the aspects, the IL has a ratio ofcation:anion of from about 2:1 to about 1:10. In some embodiments of anyof the aspects, the IL has a ratio of cation:anion of from 2:1 to 1:10.In some embodiments of any of the aspects, the IL has a ratio ofcation:anion of from about 2:1 to about 1:2. In some embodiments of anyof the aspects, the IL has a ratio of cation:anion of from 2:1 to 1:2.In some embodiments of any of the aspects, the IL has a ratio ofcation:anion such that there is a greater amount of anion, e.g., a ratioof less than 1:1. In some embodiments of any of the aspects, the IL hasa ratio of cation:anion such that there is an excess of anion. In someembodiments of any of the aspects, the IL has a ratio of cation:anion offrom about 1:1 to about 1:10. In some embodiments of any of the aspects,the IL has a ratio of cation:anion of from 1:1 to 1:10. In someembodiments of any of the aspects, the IL has a ratio of cation:anion offrom about 1:1 to about 1:4. In some embodiments of any of the aspects,the IL has a ratio of cation:anion of from 1:1 to 1:4. In someembodiments of any of the aspects, the IL has a ratio of cation:anion offrom about 1:1 to about 1:3. In some embodiments of any of the aspects,the IL has a ratio of cation:anion of from 1:1 to 1:3. In someembodiments of any of the aspects, the IL has a ratio of cation:anion offrom about 1:1 to about 1:2. In some embodiments of any of the aspects,the IL has a ratio of cation:anion of from 1:1 to 1:2. In someembodiments of any of the aspects, the IL has a ratio of cation:anion ofabout 1:1, 1:2, 1:3, or 1:4. In some embodiments of any of the aspects,the IL has a ratio of cation:anion of 1:1, 1:2, 1:3, or 1:4. Withoutwishing to be constrained by theory, compositions with higher amounts ofanion relative to cation display greater hydrophobicity.

In some embodiments of any of the aspects, the IL is at a concentrationof at least 20 mM. In some embodiments of any of the aspects, the IL isat a concentration of at least about 20 mM. In some embodiments of anyof the aspects, the IL is at a concentration of at least 25 mM. In someembodiments of any of the aspects, the IL is at a concentration of atleast about 25 mM. In some embodiments of any of the aspects, the IL isat a concentration of at least 50 mM. In some embodiments of any of theaspects, the IL is at a concentration of at least about 50 mM. In someembodiments of any of the aspects, the IL is at a concentration of atleast 100 mM, 500 mM, 1 M, 2 M, 3 M or greater. In some embodiments ofany of the aspects, the IL is at a concentration of at least about 100mM, 500 mM, 1 M, 2 M, 3 M or greater.

In some embodiments of any of the aspects, the IL is at a concentrationof from about 50 mM to about 4 M. In some embodiments of any of theaspects, the IL is at a concentration of from 50 mM to 4 M. In someembodiments of any of the aspects, the IL is at a concentration of fromabout 500 mM to about 4 M. In some embodiments of any of the aspects,the IL is at a concentration of from 500 mM to 4 M. In some embodimentsof any of the aspects, the IL is at a concentration of from about 1 M toabout 4 M. In some embodiments of any of the aspects, the IL is at aconcentration of from 1 M to 4 M. In some embodiments of any of theaspects, the IL is at a concentration of from about 2 M to about 4 M. Insome embodiments of any of the aspects, the IL is at a concentration offrom 2 M to 4 M.

In some embodiments of any of the aspects, the IL concentration in thecomposition or formulation is about 0.1 mM to 20 mM. In some embodimentsof any of the aspects, the IL concentration in the composition orformulation is about 0.5 mM to 20 mM, 0.5 mM to 18 mM, 0.5 mM to 16 mM,0.5 mM to 14 mM, 0.5 mM to 12 mM, 0.5 mM to 10 mM, 0.5 mM to 8 mM, 1 mMto 20 mM, 1 mM to 18 mM, 1 mM to 16 mM, 1 mM to 14 mM, 1 mM to 12 mM, 1mM to 10 mM, 1 mM to 8 mM, 2 mM to 20 mM, 2 mM to 18 mM, 2 mM to 16 mM,2 mM to 14 mM, 2 mM to 12 mM, 2 mM to 10 mM, 2 mM to 8 mM, 4 mM to 20mM, 4 mM to 18 mM, 4 mM to 16 mM, 4 mM to 14 mM, 4 mM to 12 mM, 4 mM to10 mM, 4 mM to 8 mM, 6 mM to 20 mM, 6 mM to 18 mM, 6 mM to 14 mM, 6 mMto 12 mM, 6 mM to 10 mM, 6 mM to 8 mM, 8 mM to 20 mM, 8 mM to 18 mM, 8mM to 16 mM, 8 mM to 14 mM, 8 mM to 12 mM, 8 mM to 10 mM, 10 mM to 20mM, 10 mM to 18 mM, 10 mM to 16 mM, 10 mM to 14 mM, 10 mM to 12 mM, 12mM to 20 mM, 12 mM to 18 mM, 12 mM to 16 mM, 12 mM to 14 mM, 14 mM to 20mM, 14 mM to 18 mM, 14 mM to 16 mM, 16 mM to 20 mM, 16 mM to 18 mM, or18 mM to 20 mM. In some embodiments of any of the aspects, the ILconcentration in the composition or formulation is about 1 mM, about 2mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM,about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about19 mM or about 20 mM.

It is specifically contemplated that a composition described herein cancomprise one, two, three, or more of any of the types of componentsdescribed herein, e.g., active agents, antifungal agents, or ILs. Forexample, a composition can comprise a mixture, solution, combination, oremulsion of two or more different ionic liquids, and/or a mixture,solution, combination, or emulsion of two or more different non-ionicsurfactants, and/or a mixture, solution, combination, or emulsion of twoor more different active compounds.

As used herein, “in combination with” refers to two or more substancesbeing present in the same formulation in any molecular or physicalarrangement, e.g, in an admixture, in a solution, in a mixture, in asuspension, in a colloid, in an emulsion. The formulation can be ahomogeneous or heterogenous mixture. In some embodiments of any of theaspects, the active compound(s) can be comprised by a superstructure,e.g., nanoparticles, liposomes, vectors, cells, scaffolds, or the like,said superstructure is which in solution, mixture, admixture,suspension, etc., with the IL.

In some embodiments of any of the aspects, the composition comprises,consists of, or consists essentially of the one or more ionic liquidscomprising comprising a quaternary ammonium cation and a undecanoic acidanion. In some embodiments of any of the aspects, the compositioncomprises, consists of, or consists essentially of choline : undecanoicacid. In some embodiments of any of the aspects, the composition doesnot comprise an active agent or ingredient other than the one or moreionic liquids comprising comprising comprising a quaternary ammoniumcation and a undecanoic acid anion. In some embodiments of any of theaspects, the composition does not comprise an active agent or ingredientother than choline : undecanoic acid. In some embodiments of any of theaspects, the composition comprises a further active agent or ingredient.

As used herein, an “active compound” or “active agent” is any agentwhich will exert an effect on a target cell or organism. The terms“compound” and “agent” refer to any entity which is normally not presentor not present at the levels being administered and/or provided to acell, tissue or subject. An agent can be selected from a groupcomprising: chemicals; small organic or inorganic molecules; signalingmolecules; nucleic acid sequences; nucleic acid analogues; proteins;peptides; enzymes; aptamers; peptidomimetic, peptide derivative, peptideanalogs, antibodies; intrabodies; biological macromolecules, extractsmade from biological materials such as bacteria, plants, fungi, oranimal cells or tissues; naturally occurring or synthetic compositionsor functional fragments thereof. In some embodiments, the agent is anychemical, entity or moiety, including without limitation synthetic andnaturally-occurring non-proteinaceous entities. Agents can be known tohave a desired activity and/or property, or can be selected from alibrary of diverse compounds. Non-limiting examples of active compoundscontemplated for use in the methods described herein include antifungalagents.

As used herein, the term “small molecule” refers to a chemical agentwhich can include, but is not limited to, a peptide, a peptidomimetic,an amino acid, an amino acid analog, a polynucleotide, a polynucleotideanalog, an aptamer, a nucleotide, a nucleotide analog, an organic orinorganic compound (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

In some embodiments of any of the aspects, the active compound can be atherapeutic compound or drug, e.g., an agent or compound which istherapeutically effective for the treatment of at least one condition ina subject. Therapeutic compounds are known in the art for a variety ofconditions, see, e.g., the database available on the world wide web atdrugs.com or the catalog of FDA-approved compounds available on theworld wide web at catalog.data.gov/dataset/drugsfda-database; each ofwhich is incorporated by reference herein in its entirety.

In some embodiments of any of the aspects, the active agent can be anantifungal agent, e.g., a further antifungal agent other than the ILdescribes herein. As used herein, the term “antifungal” refers to anycompound known to one of ordinary skill in the art that will inhibit orreduce the growth of, or kill, one or more fungal species. Thus, theability to inhibit or reduce the growth of, or kill, one or more fungalorganisms is referred to herein as “antifungal activity.” In someembodiments, an antifungal agent for use in the compositions and methodsdescribed herein is “fungistatic,” meaning that they stop fungi fromreproducing, while not necessarily harming them otherwise. Fungistaticagents limit the growth of fungi by interfering with fungi proteinproduction, DNA replication, or other aspects of fungal cellularmetabolism, and typically work together with the immune system to removefungi from the body. High concentrations of some fungistatic agents arealso fungicidal, in some cases, whereas low concentrations of somefungicidal agents are fungistatic. In some embodiments, an antifungalagent (or the effective amount thereof) for use in the compositions andmethods described herein is “fungicidal” for the target fungus. That is,the agent kills the target fungal cells and, ideally, is notsubstantially toxic to mammalian cells. Fungicidal agents includedisinfectants, and antiseptics. Many antifungal compounds are relativelysmall molecules with a molecular weight of less than 2000 atomic massunits. The term “antifungal” includes includes, but is not limited tothe antifungals described herein or any salts or variants thereof. Theantifugnal used in addition to the potentiator compound in the variousembodiments of the compositions and methods described herein will dependon the type of fungal infection.

Major classes of known antifungal agents include, for example, polyenes,imidazoles, triazoles, thiazoles, allylamine, and echinocandins.Accordingly, non-limiting examples of antifungal agents that aresuitable for use with the compositions and methods described hereininclude, without limitation, amphotericin B, candicidin, filipin,hamycin, natamycin, nystatin, rimocidin, bifonazole, butoconazole,clotrimazole, econazole, fenticonzole, isoconazole, ketoconazole,miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole,tioconazole, albaconazole; fluconazole; isavuconazole; itraconazole;posaconazole; ravuconazole; terconazole; voriconazole, abafungin,amorolfin; butenafine; naftifine; terbinafine, anidulafungin;caspofungin; and micafungin.

Antifungal polyenes are macrocyclic polyenes with a heavily hydroxylatedregion on the ring opposite the conjugated system, rendering themamphiphilic. Polyenes act by binding to sterols, e.g. ergosterol, in thefungal membrane, making the membrane more crystalline. The polyene,amphotericin B (AMB), introduced in the late 1950s, was the first widelyutilized antifungal (AF) drug. Due to its strong hydrophobicity, AMBpenetrates the fungal membrane and binds to ergosterol leading tomembrane damage. Non-limiting examples of polyenes can includeamphotericin B; candicidin; filipin; hamycin; natamycin; nystatin; andrimocidin.

Azoles inhibit ergosterol biosynthesis and lead to the accumulation of atoxic methylated sterol that stops cell growth. While azoles tend to befungistatic due to their poor solubility, under certain conditions andformulations, azoles such as miconazole (MCZ) can be fungicidal.Non-limiting examples of imidazoles can include bifonazole;butoconazole; clotrimazole; econazole; fenticonzole; isoconazole;ketoconazole; miconazole; omoconazole; oxiconazole; sertaconazole;sulconazole; and tioconazole. Non-limiting examples of triazoles caninclude albaconazole; fluconazole; isavuconazole; itraconazole;posaconazole; ravuconazole; terconazole; and voriconazole. In someembodiments, the antifungal agent can be a thiazole, e.g. abafungin.

Echinocandins inhibit the synthesis of cell wall glucan. Non-limitingexamples of echinocandins can include anidulafungin; caspofungin; andmicafungin.

Allylamines inhibit squalene epoxidase, which is required for ergosterolbiosynthesis. Non-limiting examples of allylamines can includeamorolfin; butenafine; naftifine; and terbinafine

Further non-limiting examples of antifungal agents can include benzoicacid; ciclopirox; flucytosine; griseofulvin; haloprogin; polygodial;tolnaftate; undecylenic acid; and crystal violet.

In some embodiments of any of the aspects, an antifungal agent can beselected from the group consisting of amphotericin B; natamycin;voriconazole; povidone-iodine; hypochlorous acid; Chlorhexidinedigluconate (CDG); vancomycin (VAN); chloramphenicol (CHL); polymyxin B(PMB); trimethoprim (TMP); benzalkonium chloride (BAK); and combinationsthereof.

In some embodiments of any of the aspects, the active compound is anantibody or antibody reagent. As used herein, the term “antibodyreagent” refers to a polypeptide that includes at least oneimmunoglobulin variable domain or immunoglobulin variable domainsequence and which specifically binds a given antigen. An antibodyreagent can comprise an antibody or a polypeptide comprising anantigen-binding domain of an antibody. In some embodiments, an antibodyreagent can comprise a monoclonal antibody or a polypeptide comprisingan antigen-binding domain of a monoclonal antibody. For example, anantibody can include a heavy (H) chain variable region (abbreviatedherein as VH), and a light (L) chain variable region (abbreviated hereinas VL). In another example, an antibody includes two heavy (H) chainvariable regions and two light (L) chain variable regions. The term“antibody reagent” encompasses antigen-binding fragments of antibodies(e.g., single chain antibodies, Fab and sFab fragments, F(ab′)2, Fdfragments, Fv fragments, scFv, and domain antibodies (dAb) fragments aswell as complete antibodies.

In some embodiments of any of the aspects, a composition as describedherein, comprising at least one IL and optionally an active compound canbe formulated as an oral, subcutaneous, intravenous, intradermal,ocular, or parenteral formulation. In some embodiments of any of theaspects, the composition described herein can be formulated for ocularadministration.

In some embodiments of any of the aspects, described herein is acomposition comprising at least one IL as described herein and at leastone active compound. In some embodiments of any of the aspects,described herein is a composition consisting essentially of at least oneIL as described herein and at least one active compound. In someembodiments of any of the aspects, described herein is a compositionconsisiting of at least one IL as described herein and at least oneactive compound. In some embodiments of any of the apsects, thecomposition comprising at least one IL as described herein and at leastone active compound is administered as a monotherapy, e.g., anothertreatment for the condition is not administered to the subject.

In one aspect of any of the embodiments, described herein is apharmaceutical composition comprising at least one IL as describedherein, and optionally at least one further active agent. In someembodiments, the pharmaceutical composition comprises the at least oneIL as described herein. In some embodiments, the pharmaceuticalcomposition consists essentially of the at least one IL as describedherein. In some embodiments, the pharmaceutical composition consists ofthe at least one IL as described herein. In some embodiments, thepharmaceutical composition consists essentially of an aqeous solution ofthe at least one IL as described herein. In some embodiments, thepharmaceutical composition consists of an aqeous solution of the atleast one IL as described herein.

In some embodiments, the pharmaceutical composition comprises the atleast one IL and the one or more active compounds as described herein.In some embodiments, the pharmaceutical composition consists essentiallyof the at least one IL and the one or more active compounds as describedherein. In some embodiments, the pharmaceutical composition consists ofthe at least one IL and the one or more active compounds as describedherein. In some embodiments, the pharmaceutical composition consistsessentially of an aqeous solution of the at least one IL and the one ormore active compounds as described herein. In some embodiments, thepharmaceutical composition consists of an aqeous solution of the atleast one IL and the one or more active compounds as described herein.

The compositions described herein can comprise at least one IL asdescribed herein, e.g., one IL, two ILs, three ILs, or more. In someembodiments of any of the aspects a composition as described herein cancomprise at least one IL as described herein and CAGE (Choline AndGEranate).

In some embodiments of any of the apsects, the composition comprising atleast one ionic liquid as described herein further comprises at leastone non-ionic surfactant. As used herein, “non-ionic surfactant” refersto a surfactant which lacks a net ionic charge and does not dissociateto an appreciable extent in aqueous media. The properties of non-ionicsurfactants are largely dependent upon the proportions of thehydrophilic and hydrophobic groups in the molecule. Hydrophilic groupsinclude the oxyethylene group (—OCH2 CH2—) and the hydroxy group. Byvarying the number of these groups in a hydrophobic molecule, such as afatty acid, substances are obtained which range from stronglyhydrophobic and water insoluble compounds, such as glycerylmonostearate, to strongly hydrophilic and water-soluble compounds, suchas the macrogols. Between these two extremes types include those inwhich the proportions of the hydrophilic and hydrophobic groups are moreevenly balanced, such as the macrogol esters and ethers and sorbitanderivatives. Suitable non-ionic surfactants may be found in Martindale,The Extra Pharmacopoeia, 28th Edition, 1982, The Pharmaceutical Press,London, Great Britain, pp. 370 to 379. Non-limiting examples ofnon-ionic surfactants include polysorbates, a Tween™, block copolymersof ethylene oxide and propylene oxide, glycol and glyceryl esters offatty acids and their derivatives, polyoxyethylene esters of fatty acids(macrogol esters), polyoxyethylene ethers of fatty acids and theirderivatives (macrogol ethers), polyvinyl alcohols, and sorbitan esters,sorbitan monoesters, ethers formed from fatty alcohols and polyethyleneglycol, polyoxyethylene-polypropylene glycol, alkyl polyglycoside,Cetomacrogol 1000, cetostearyl alcohol, cetyl alcohol, cocamide DEA,cocamide MEA, decyl glucoside, decyl polyglucose, glycerol monostearate,IGEPAL CA-630, isoceteth-20, lauryl glucoside, maltosides, monolaurin,mycosubtilin, Nonidet P-40, nonoxynol-9, nonoxynols, NP-40, octaethyleneglycol monododecyl ether, N-Octyl beta-D-thioglucopyranoside, octylglucoside, oleyl alcohol, PEG-10 sunflower glycerides, pentaethyleneglycol monododecyl ether, polidocanol, poloxamer, poloxamer 407,polyethoxylated tallow amine, polyglycerol polyricinoleate, sorbitan,sorbitan monolaurate, sorbitan monostearate, sorbitan tristearate,stearyl alcohol, surfactin, Triton X-100, and the like. In someembodiments of any of the aspects, the at least one non-ionic surfactanthas a neutral hydrophilic head group.

As used herein, “polysorbate” refers to a surfactant derived fromethoxylated sorbitan (a derivative of sorbitol) esterified with fattyacids. Common brand names for polysorbates include Scattics™, Alkest™,Canarcel™, and Tween™. Exemplary polysorbates include polysorbate 20(polyoxyethylene (20) sorbitan monolaurate), polysorbate 40(polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60(polyoxyethylene (20) sorbitan monostearate), and polysorbate 80(polyoxyethylene (20) sorbitan monooleate).

In some embodiments of any of the aspects, the at least one non-ionicsurfactant (e.g., at least one polysorbate) is present at aconcentration of about 0.1% to about 50% w/v. In some embodiments of anyof the aspects, the at least one non-ionic surfactant (e.g., at leastone polysorbate) is present at a concentration of 0.1% to 50% w/v. Insome embodiments of any of the aspects, the at least one non-ionicsurfactant (e.g., at least one polysorbate) is present at aconcentration of about 1% to about 5% w/v. In some embodiments of any ofthe aspects, the at least one non-ionic surfactant (e.g., at least onepolysorbate) is present at a concentration of 1% to 5% w/v. In someembodiments of any of the aspects, the at least one non-ionic surfactant(e.g., at least one polysorbate) is present at a concentration of about3% to about 10% w/v. In some embodiments of any of the aspects, the atleast one non-ionic surfactant (e.g., at least one polysorbate) ispresent at a concentration of 3% to 10% w/v. In some embodiments of anyof the aspects, the at least one non-ionic surfactant (e.g., at leastone polysorbate) is present at a concentration of less than about 5%w/v. In some embodiments of any of the aspects, the at least onenon-ionic surfactant (e.g., at least one polysorbate) is present at aconcentration of less than 5% w/v.

In some embodiments of any of the aspects, the at least one IL asdescribed herein, and optionally any further active agents, is providedin one or more nanoparticles. In some embodiments of any of the aspects,the composition comprising the at least one IL as described herein, andoptionally any further active agents, comprises nanoparticles comprisingthe active agent, e.g., the nanoparticles are in solution or suspensionin a composition comprising at least one IL as described herein.

In some embodiments of any of the aspects, a composition as describedherein, e.g., a composition comprising at least one IL and optionallyany further active agents, can further comprise a pharmaceuticallyacceptable carrier. As used herein, the terms “pharmaceuticallyacceptable”, “physiologically tolerable” and grammatical variationsthereof, as they refer to compositions, carriers, diluents and reagents,are used interchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike. A pharmaceutically acceptable carrier will not promote the raisingof an immune response to an agent with which it is admixed, unless sodesired. The preparation of a pharmacological composition that containsactive ingredients dissolved or dispersed therein is well understood inthe art and need not be limited based on formulation. Typically, suchcompositions are prepared as injectable either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified or presented as a liposome composition. The active ingredientcan be mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient and in amounts suitable for use inthe therapeutic methods described herein. Suitable excipients include,for example, water, saline, dextrose, glycerol, ethanol or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents and the like which enhance theeffectiveness of the active ingredient. The therapeutic composition ofthe present disclosure can include pharmaceutically acceptable salts ofthe components therein. Pharmaceutically acceptable salts include theacid addition salts (formed with the free amino groups of thepolypeptide) that are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,tartaric, mandelic and the like. Salts formed with the free carboxylgroups can also be derived from inorganic bases such as, for example,sodium, potassium, ammonium, calcium or ferric hydroxides, and suchorganic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine and the like. Physiologically tolerable carriers arewell known in the art. Exemplary liquid carriers are sterile aqueoussolutions that contain no materials in addition to the activeingredients and water, or contain a buffer such as sodium phosphate atphysiological pH value, physiological saline or both, such asphosphate-buffered saline. Still further, aqueous carriers can containmore than one buffer salt, as well as salts such as sodium and potassiumchlorides, dextrose, polyethylene glycol and other solutes. Liquidcompositions can also contain liquid phases in addition to and to theexclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions. The amount of an active agent used in the methods describedherein that will be effective in the treatment of a particular disorderor condition will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques. Suitablepharmaceutical carriers are described in Remington’s PharmaceuticalSciences, A. Osol, a standard reference text in this field of art. Forexample, a parenteral composition suitable for administration byinjection is prepared by dissolving 1.5% by weight of active ingredientin 0.9% sodium chloride solution.

The term “carrier” in the context of a pharmaceutical carrier refers toa diluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations, and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in Remington’sPharmaceutical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing Co.,1990). The formulation should suit the mode of administration.

Pharmaceutically acceptable carriers and diluents include saline,aqueous buffer solutions, solvents and/or dispersion media. The use ofsuch carriers and diluents is well known in the art. Some non-limitingexamples of materials which can serve as pharmaceutically-acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, methylcellulose,ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, suchas magnesium stearate, sodium lauryl sulfate and talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer’s solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein. In someembodiments, the carrier inhibits the degradation of the activecompound. The term “pharmaceutically acceptable carrier” excludes tissueculture medium.

In some embodiments of any of the aspects described herein, thebiological activity of an active agent is improved or stabilized ascompared to the activity in the absence of the at least one IL.

The compositions described herein, e.g., those comprising at least oneIL comprising comprising a quaternary ammonium cation and a undecanoicacid anion are demonstrated to have surprisingly efficacious antifungalactivity coupled with low toxicity. Accordingly, in one aspect of any ofthe embodiments, described herein is a method of treating or preventinga fungal infection, comprising administering to a subject in needthereof a composition of any of the preceding claims. In someembodiments of any of the aspects, the fungal infection is an ocularfungal infection and the composition is administered to one or botheyes. In some embodiments of any of the aspects, the subject is one whohas received a corneal surgery.

Subjects receiving corneal surgery, e.g., keratoprosthesis (KPro)implantation, artificial cornea surgery, or cornea replacement surgeryare particularly suspectible to later development of ocular infections.Such subjects frequently receive prophylactic treatments to prevent orslow ocular infections. Accordingly, in some embodiments of any of theaspects, the method further comprises a first step of performing cornealsurgery on one or both eyes of the subject. In one aspect of any of theembodiments, described herein is a method comprising performing cornealsurgery on one or both eyes of a subject and then administering acomposition as described herein, e.g, comprising at least one ILcomprising comprising a quaternary ammonium cation and a undecanoic acidanion to the one or both eyes. In some embodiments of any of theaspects, the corneal surgery is keratoprosthesis (KPro) implantation,artificial cornea surgery, or cornea replacement surgery.

The methods described herein can, in some aspects and embodiments, beused to inhibit, delay formation of, treat, and/or prevent or provideprophylactic treatment of fungal infections in animals, includinghumans.

In some embodiments of any of the aspects, described herein is aprophylactic method of treatment. As used herein “prophylactic” refersto the timing and intent of a treatment relative to a disease orsymptom, that is, the treatment is administered prior to clinicaldetection or diagnosis of that particular disease or symptom in order toprotect the patient from the disease or symptom. Prophylactic treatmentcan encompass a reduction in the severity or speed of onset of thedisease or symptom, or contribute to faster recovery from the disease orsymptom. Accordingly, the methods described herein can be prophylacticrelative to a fungal infection, increase in a fungal infection, ornegative effects on the eye from a fungal infection. In some embodimentsof any of the aspects, prophylactic treatment is not prevention of allsymptoms or signs of a disease.

As used herein, a “fungal infection” refers to an abnormal and/orundesired presence of a fungus in or on a subject. The presence can beabnormal in that the fungus is a noncommensal species, e.g. one nottypically found in or on a healthy subject, or it can be abnormal inthat the fungus is present at at abnormally high levels, e.g. at leasttwice the level found in or on a healthy subject (e.g. twice the level,three times the level, four times the level, five times the level, orgreater), or it can be abnormal in that the presence of the fungus iscausing or contributing to disease or symptoms thereof, e.g. necrosis,disfigurement, delayed wound healing, etc.

In some embodiments, the methods described herein relate to treating asubject having or diagnosed as having a condition with a composition asdescribed herein, e.g, a comprising at least one IL as described hereinand and optionally at least one further active agents. Subjects having acondition, e.g., a fungal infection, can be identified by a physicianusing current methods of diagnosing fungal infections. Diagnosis can beby any suitable means. Diagnosis and monitoring can involve, forexample, detecting the level of fungal load in a biological sample (forexample, a tissue biopsy, blood test, or urine test), detecting thelevel of a surrogate marker of the fungal infection in a biologicalsample, detecting symptoms associated with the infection, or detectingimmune cells involved in the immune response typical of fungalinfections (for example, detection of antigen specific T cells orantibody production). In some embodiments of any of the aspects, thesubject is identified as having a fungal infection by objectivedetermination of the presence of fungal cells in the subject’s body byone of skill in the art. Such objective determinations can be performedthrough the sole or combined use of tissue analyses, blood analyses,urine analyses, and fungal cell cultures, in addition to the monitoringof specific symptoms associated with the fungal infection.

Examples of fungal infections include but are not limited to:aspergillosis; thrush (caused by Candida albicans); cryptococcosis(caused by Cryptococcus); and histoplasmosis. Thus, examples ofinfectious fungi include, but are not limited to, Cryptococcusneoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomycesdermatitidis, Chlamydia trachomatis, Candida albicans. Furthernon-limiting examples of fungal infections include Candida spp.;Cryptococcus spp.; Aspergillus spp.; Microsporum spp.; Trichophytonspp.; Epidermophyton spp.; Trichosporon spp.; Fusarium spp.; Tineaversicolor; Tinea barbae; Tinea corporis; Tinea cruris; Tinea manuum;Tinea pedis; Tinea unguium; Tinea faciei; Tinea imbricate; Tineaincognito; Epidermophyton floccosum; Microsporum canis; Microsporumaudouinii; Trichophyton interdigitale; Trichophyton mentagrophytes;Trichophyton tonsurans; Trichophyton schoenleini; Trichophyton rubrum;Hortaea werneckii; Piedraia hortae; Malasserzia furfur; Coccidioidesimmitis; Coccidioides posadasii; Histoplasma capsulatum; Histoplasmaduboisii; Lacazia loboi; Paracoccidioides brasiliensis; Blastomycesdermatitidis; Sporothrix schenckii; Penicillium marneffei; Candidaalbicans; Candida glabrata; Candida tropicalis; Candida lusitaniae;Candida jirovecii; Candida krusei; Candida parapsilosi; Exophialajeanselmei; Fonsecaea pedrosoi; Fonsecasea compacta; Phialophoraverrucosa; Geotrichum candidum; Pseudallescheria boydii; Rhizopusoryzae; Muco indicus; Absidia corymbifera; Synceplasastrum racemosum;Basidiobolus ranarum; Conidiobolus coronatus; Conidiobolus incongruous;Cryptococcus neoformans; Enterocytozoan bieneusi; Encephalitozoonintestinalis; and Rhinosporidium seeberi. In some embodiments of any ofthe aspects, the fungal infection is infectious keratitis and/orendopthalmitis. In some embodiments of any of the aspects, the fungalinfection is an ocular fungal infection. In some embodiments of any ofthe aspects, the fungal infection is an infection of Candida, Candidaalbicans, Candida parapsilosis, Fusarium, and/or Aspergillus. Thecompositions and methods described herein are contemplated for use intreating infections with these and other fungi.

The compositions and methods described herein can be administered to asubject having or diagnosed as having a condition described herein. Insome embodiments, the methods described herein comprise administering aneffective amount of compositions described herein, e.g. a compositoncomprising at least one IL as described herein and optionally at leastone further active agent, to a subject in order to alleviate a symptomof a condition described herein. As used herein, “alleviating a symptom”is ameliorating any marker or symptom associated with a condition. Ascompared with an equivalent untreated control, such reduction is by atleast 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more asmeasured by any standard technique. A variety of means for administeringthe compositions described herein to subjects are known to those ofskill in the art. Such methods can include, but are not limited to oral,parenteral, intravenous, intramuscular, subcutaneous, transdermal,airway (aerosol), pulmonary, cutaneous, injection, or intratumoraladministration. Administration can be local or systemic.

In some embodiments of any of the aspects, the administration istransdermal. In some embodiments of any of the aspects, theadministration is transdermal, to a mucus membrane (e.g., to a nasal,oral, or vaginal membrane), oral, subcutaneous, intradermal, parenteral,intratumoral, ocular, corneally, or intravenous. In some embodiments ofany of the aspects, the administration is provided in or on a contactlens, a lower conjunctival fornix device, or a subconjunctival device.

Formulations for ocular delivery, e.g., ophthalmic delivery can be usedin the compositions and methods described herein. Such formulations cangenerally comprise an admixture of the compositions described hereinwith an ophthalmically acceptable vehicle. An “ophthalmically acceptablevehicle” is one having physical properties (e.g., pH and/or osmolality)that are physiologically compatible with ophthalmic tissues, e.g., theretina, among others.

In some embodiments of any of the aspects, an ophthalmic composition isformulated as a sterile aqueous solution having an osmolality of fromabout 200 to about 400 milliosmoles/kilogram water (“mOsm/kg”) and aphysiologically compatible pH. The osmolality of the solutions can beadjusted, for example, by means of conventional agents, such asinorganic salts (e.g., NaCl), organic salts (e.g., sodium citrate),polyhydric alcohols (e.g., propylene glycol or sorbitol) or combinationsthereof.

Ophthalmic formulations can be in the form of liquid, solid or semisoliddosage form. Ophthalmic formulations can comprise, depending on thefinal dosage form, suitable ophthalmically acceptable excipients. Insome embodiments, ophthalmic formulations are formulated to maintain aphysiologically tolerable pH range. In certain embodiments, the pH rangeof an ophthalmic formulation is in the range of from about 5 to about 9.In some embodiments, pH of an ophthalmic formulation is in the range offrom about 6 to about 8, or is about 6.5, about 7, or about 7.5. One ormore ophthalmically acceptable pH adjusting agents and/or bufferingagents can be included in a composition for ophthalmic delivery,including acids such as acetic, boric, citric, lactic, phosphoric, andhydrochloric acids; bases such as sodium hydroxide, sodium phosphate,sodium borate, sodium citrate, sodium acetate, and sodium lactate; andbuffers such as citrate/dextrose, sodium bicarbonate, and ammoniumchloride. Such acids, bases, and buffers can be included in an amountrequired to maintain pH of the composition in an ophthalmicallyacceptable range. One or more ophthalmically acceptable salts can beincluded in the composition in an amount sufficient to bring osmolalityof the composition into an ophthalmically acceptable range. Such saltsinclude those having sodium, potassium, or ammonium cations andchloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate,thiosulfate, or bisulfite anions.

In some embodiments of any of the aspects, a composition for ophthalmicdelivery can be for topical delivery, e.g., in the form of an eye drop.By means of a suitable dispenser, a desired dosage of the active agentcan be metered by administration of a known number of drops into theeye, such as by one, two, three, four, or five drops. Additional ocularpharmaceutical compositions and delivery devices are further described,e.g., in U.S. Pat. Nos. 9,993,558 B2; 4,310,543A; 8,668,676 B2, and4,853,224 A, the contents of each of which are incorporated herein byreference in their entireties.

In some embodiments of any of the aspects, a composition describedherein is provided in or on a lower conjunctival fornix device, or asubconjunctival device. Such devices can be implantation devices ordiposable devices that can be placed in or on the indicated locationwhere they release the composition, e.g., by diffusion.

Oral administration can comprise providing tablets (including withoutlimitation scored or coated tablets), pills, caplets, capsules, chewabletablets, powder packets, cachets, troches, wafers, aerosol sprays, orliquids, such as but not limited to, syrups, elixirs, solutions orsuspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-wateremulsion, or a water-in-oil emulsion. Oral formulations can comprisediscrete dosage forms, such as, but not limited to, tablets (includingwithout limitation scored or coated tablets), pills, caplets, capsules,chewable tablets, powder packets, cachets, troches, wafers, aerosolsprays, or liquids, such as but not limited to, syrups, elixirs,solutions or suspensions in an aqueous liquid, a non-aqueous liquid, anoil-in-water emulsion, or a water-in-oil emulsion. Such compositionscontain a predetermined amount of an ionic liquid as described hereinand the at least one active compound, and may be prepared by methods ofpharmacy well known to those skilled in the art. See generally,Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott,Williams, and Wilkins, Philadelphia PA. (2005).

In some embodiments of any of the aspects, subcutaneous, intradermal orintravenous administration comprises administration via injection,catheter, port, or the like.

In some embodiments of any of the aspects, the composition describedherein can be a parenteral dose form. Since administration of parenteraldosage forms typically bypasses the patient’s natural defenses againstcontaminants, parenteral dosage forms are preferably sterile or capableof being sterilized prior to administration to a patient. Examples ofparenteral dosage forms include, but are not limited to, solutions readyfor injection, dry products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions. In addition, controlled-release parenteraldosage forms can be prepared for administration of a patient, including,but not limited to, DUROS®-type dosage forms and dose-dumping.

Suitable vehicles that can be used to provide parenteral dosage forms ofa composition comprising an ionic liquid as described herein are wellknown to those skilled in the art. Examples include, without limitation:sterile water; water for injection USP; saline solution; glucosesolution; aqueous vehicles such as but not limited to, sodium chlorideinjection, Ringer’s injection, dextrose Injection, dextrose and sodiumchloride injection, and lactated Ringer’s injection; water-misciblevehicles such as, but not limited to, ethyl alcohol, polyethyleneglycol, and propylene glycol; and non-aqueous vehicles such as, but notlimited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyloleate, isopropyl myristate, and benzyl benzoate. Compounds that alteror modify the solubility of an ingredient in a composition as disclosedherein can also be incorporated into the parenteral dosage forms of thedisclosure, including conventional and controlled-release parenteraldosage forms.

Conventional dosage forms generally provide rapid or immediate drugrelease from the formulation. Depending on the pharmacology andpharmacokinetics of the drug, use of conventional dosage forms can leadto wide fluctuations in the concentrations of the drug in a patient’sblood and other tissues. These fluctuations can impact a number ofparameters, such as dose frequency, onset of action, duration ofefficacy, maintenance of therapeutic blood levels, toxicity, sideeffects, and the like. While as noted above herein, the compositions asdescribed herein can obviate certain reasons for using acontrolled-release formulation, it is contemplated herein that themethods and compositions can be utilized in controlled-releaseformulations in some embodiments. For example, controlled-releaseformulations can be used to control a drug’s onset of action, durationof action, plasma levels within the therapeutic window, and peak bloodlevels. In particular, controlled- or extended-release dosage forms orformulations can be used to ensure that the maximum effectiveness of adrug is achieved while minimizing potential adverse effects and safetyconcerns, which can occur both from under-dosing a drug (i.e., goingbelow the minimum therapeutic levels) as well as exceeding the toxicitylevel for the drug. In some embodiments, the composition as describedherein can be administered in a sustained release formulation.

Controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledrelease counterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include: 1) extended activity of the drug; 2) reduceddosage frequency; 3) increased patient compliance; 4) usage of lesstotal drug; 5) reduction in local or systemic side effects; 6)minimization of drug accumulation; 7) reduction in blood levelfluctuations; 8) improvement in efficacy of treatment; 9) reduction ofpotentiation or loss of drug activity; and 10) improvement in speed ofcontrol of diseases or conditions. Kim, Cherng-ju, Controlled ReleaseDosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release other amountsof drug to maintain this level of therapeutic or prophylactic effectover an extended period of time. In order to maintain this constantlevel of drug in the body, the drug must be released from the dosageform at a rate that will replace the amount of drug being metabolizedand excreted from the body. Controlled-release of an active ingredientcan be stimulated by various conditions including, but not limited to,pH, ionic strength, osmotic pressure, temperature, enzymes, water, andother physiological conditions or compounds.

A variety of known controlled- or extended-release dosage forms,formulations, and devices can be adapted for use with the salts andcompositions of the disclosure. Examples include, but are not limitedto, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809;3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591,767; 5,120,548;5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1; each ofwhich is incorporated herein by reference. These dosage forms can beused to provide slow or controlled-release of one or more activeingredients using, for example, hydroxypropylmethyl cellulose, otherpolymer matrices, gels, permeable membranes, osmotic systems (such asOROS® (Alza Corporation, Mountain View, Calif. USA)), or a combinationthereof to provide the desired release profile in varying proportions.

The term “effective amount” as used herein refers to the amount of acomposition needed to alleviate at least one or more symptom of thedisease or disorder, and relates to a sufficient amount ofpharmacological composition to provide the desired effect. The term“therapeutically effective amount” therefore refers to an amount of acomposition that is sufficient to provide a particular effect whenadministered to a typical subject. An effective amount as used herein,in various contexts, would also include an amount sufficient to delaythe development of a symptom of the disease, alter the course of asymptom disease (for example but not limited to, slowing the progressionof a symptom of the disease), or reverse a symptom of the disease. Thus,it is not generally practicable to specify an exact “effective amount”.However, for any given case, an appropriate “effective amount” can bedetermined by one of ordinary skill in the art using only routineexperimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dosage can vary depending upon the dosage formemployed and the route of administration utilized. The dose ratiobetween toxic and therapeutic effects is the therapeutic index and canbe expressed as the ratio LD50/ED50. Compositions and methods thatexhibit large therapeutic indices are preferred. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC50 (i.e., theconcentration of the active compound, which achieves a half-maximalinhibition of symptoms) as determined in cell culture, or in anappropriate animal model. Levels in plasma can be measured, for example,by high performance liquid chromatography. The effects of any particulardosage can be monitored by a suitable bioassay, e.g., assay for bloodglucose, among others. The dosage can be determined by a physician andadjusted, as necessary, to suit observed effects of the treatment.

In some embodiments of any of the apsects, the composition as describedherein, e.g., a composition comprising at least one IL as describedherein is administered as a monotherapy, e.g., another treatment for thecondition is not administered to the subject.

In some embodiments of any of the aspects, the methods described hereincan further comprise administering a second agent and/or treatment tothe subject, e.g. as part of a combinatorial therapy, either in thecomposition described herein, e.g., a composition comprising at leastone IL as described herein, or as a separate formulation. For example,non-limiting examples of a second agent and/or treatment for the subjectdescribed herein include further antifungal agents as describedelsewhere herein as well as antibiotic agents to prevent bacterialinfections.

As used herein, “antibiotic” refers to any chemical or biological agentwith therapeutic usefulness in the inhibition of bacterial cell growthor in killing bacteria, e.g, those that are bactericidal orbacteriostatic. Categories of antibiotics can include, but are notlimited to those that target the bacterial cell wall (e.g., penicillins,cephalosporins), those that target the bacterial cell membrane (e.g.,polymyxins), those that target bacterial enzymes (e.g., rifamycins,lipiarmycins, quinolones, sulfonamides), protein synthesis inhibitors(e.g., macrolides, lincosamides, and tetracyclines), aminoglycosides,cyclic lipopeptides, glycyclines, oxazolidinones, beta-lactams, andlipiarmycins. Exemplary, non-limiting antibiotics include penicillin,methicilling, nafcillin, oxacillin, cloxacillin, dicloxacillin,flucloxacillin, ampicillin, amoxicillin, pivampicillin, hetacillin,bacampicillin, metampicillin, talamipicillin, epicillin, cabenicillin,ticaricillin, temocillin, mezlocillin, piperacillin, azolocillin,clavulanic acid, sulbactam, tazobactam, cafadroxil, cephalexin,cefalotin, cefapirin, cefazolin, cefradine, cefaclor, cefonicid,cefprozil, cefuroxime, loracarbef, cefmetazole, cefotetan, cefoxitin,cefotiam, cefdinir, cefixime, cefotaxime, cefovecin, cefpodoxime,ceftibuten, ceftiofur, ceftizoxime, ceftriaxone, cefoperazone,ceftazimdime, latamoxef, cefepime, cefiderocol, cefpriome, rifampicin,rifabutin, rifapentine, rifamixin, fidaxomicin, ciproflaxicin,moxifloxacin, levofloxacin, sulfafurzole, azithromycin, clarithromycin,erythromycin, fidaxomicin, spiramycin, telihtromycin, lincomycin,clindamycin, pirlimycin, tetracycline, eravacycline, sarecycline,omadacycline, doxycycline, kanamycin, tobramycin, gentamicin, neomycin,streptomycin, vancomycin, tigecycline, linezolid, posizolid, tedizolid,radezolid, cycloserine, contezolid, and daptomycin. In some embodimentsof any of the aspects, the antibiotic is one or more of tobramycin,trimethroprim, ciprofloxacin, gatifloxacin, moxifloxacin,fluoroguinolones, cefazolin, and vancomycin. One of skill in the art canreadily identify an antibiotic agent of use e.g. see Antibiotics inLaboratory Medicine, Victor Lorian (ed.) Wolters Kluwer; and AntiboticsManual, David Schlossberg and Rafik Samuel, John Wiley and Sons (2017);each of which is incorporated by reference herein in its entirety.

In certain embodiments, an effective dose of a composition describedherein, e.g, a composition comprising at least one IL as describedherein, can be administered to a patient once. In certain embodiments,an effective dose a composition described herein, e.g, a compositioncomprising at least one IL as described herein, can be administered to apatient repeatedly. In certain embodiments, an effective dose acomposition described herein, e.g, a composition comprising at least oneIL as described herein, can be administered to a patient daily. Forsystemic administration, subjects can be administered a therapeuticamount of a composition described herein, e.g, a composition comprisingat least one IL as described herein, such as, e.g. 0.1 mg/kg, 0.5 mg/kg,1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg,25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more. In some embodiments ofany of the aspects, the at least one IL is present at a dose of from1.0-20.0 mg/kg. In some embodiments of any of the aspects, the at leastone IL is present at a dose fo from about 1.0- about 20.0 mg/kg.

The dosage of a composition as described herein can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment. With respect to duration and frequency of treatment, it istypical for skilled clinicians to monitor subjects in order to determinewhen the treatment is providing therapeutic benefit, and to determinewhether to increase or decrease dosage, increase or decreaseadministration frequency, discontinue treatment, resume treatment, ormake other alterations to the treatment regimen. The dosing schedule canvary from once a week to daily depending on a number of clinicalfactors, such as the subject’s sensitivity to the active compound. Thedesired dose or amount of activation can be administered at one time ordivided into subdoses, e.g., 2-4 subdoses and administered over a periodof time, e.g., at appropriate intervals through the day or otherappropriate schedule. In some embodiments, administration can bechronic, e.g., one or more doses and/or treatments daily over a periodof weeks or months. Examples of dosing and/or treatment schedules areadministration daily, twice daily, three times daily or four or moretimes daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month,2 months, 3 months, 4 months, 5 months, or 6 months, or more. Acomposition described herein, e.g, a composition comprising at least oneIL as described herein, can be administered over a period of time, suchas over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minuteperiod.

The dosage ranges for the administration of the compositions describedherein, according to the methods described herein depend upon, forexample, the form of the active compound, its potency, and the extent towhich symptoms, markers, or indicators of a condition described hereinare desired to be reduced, for example the percentage reduction desiredfor symptoms or markers. The dosage should not be so large as to causeadverse side effects. Generally, the dosage will vary with the age,condition, and sex of the patient and can be determined by one of skillin the art. The dosage can also be adjusted by the individual physicianin the event of any complication.

The efficacy of a composition described in, e.g. the treatment of acondition described herein, or to induce a response as described hereincan be determined by the skilled clinician. However, a treatment isconsidered “effective treatment,” as the term is used herein, if one ormore of the signs or symptoms of a condition described herein arealtered in a beneficial manner, other clinically accepted symptoms areimproved, or even ameliorated, or a desired response is induced e.g., byat least 10% following treatment according to the methods describedherein. Efficacy can be assessed, for example, by measuring a marker,indicator, symptom, and/or the incidence of a condition treatedaccording to the methods described herein or any other measurableparameter appropriate. Efficacy can also be measured by a failure of anindividual to worsen as assessed by hospitalization, or need for medicalinterventions (i.e., progression of the disease is halted). Methods ofmeasuring these indicators are known to those of skill in the art and/orare described herein. Treatment includes any treatment of a disease inan individual or an animal (some non-limiting examples include a humanor an animal) and includes: (1) inhibiting the disease, e.g., preventinga worsening of symptoms (e.g. pain or inflammation); or (2) relievingthe severity of the disease, e.g., causing regression of symptoms. Aneffective amount for the treatment of a disease means that amount which,when administered to a subject in need thereof, is sufficient to resultin effective treatment as that term is defined herein, for that disease.Efficacy of an agent can be determined by assessing physical indicatorsof a condition or desired response. It is well within the ability of oneskilled in the art to monitor efficacy of administration and/ortreatment by measuring any one of such parameters, or any combination ofparameters. Efficacy can be assessed in animal models of a conditiondescribed herein, for example treatment of diabetes or cancer. Whenusing an experimental animal model, efficacy of treatment is evidencedwhen a statistically significant change in a marker is observed.

In vitro and animal model assays are provided herein which allow theassessment of a given dose of a composition described herein, e.g, acomposition comprising at least one IL as described herein. By way ofnon-limiting example, the effects of a dose of a composition comprisingat least one IL as described can be assessed by using the modelsdescribed in the Examples herein, e.g, fungal growth in vitro and/or ina corneal model.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

A carboxylic acid is a carbonyl-bearing functional group having aformula RCOOH where R is aliphatic, heteroaliphatic, alkyl, orheteroalkyl.

As used herein, the term “alkyl” means a straight or branched, saturatedaliphatic radical having a chain of carbon atoms. The term “alkyl”includes cycloalkyl or cyclic alkyl. C_(x) alkyl and C_(x)-C_(y)alkylare typically used where X and Y indicate the number of carbon atoms inthe chain. For example, C₁-C₆alkyl includes alkyls that have a chain ofbetween 1 and 6 carbons (e.g., methyl, ethyl, propyl, isopropyl, butyl,sec-butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and thelike). Alkyl represented along with another radical (e.g., as inarylalkyl) means a straight or branched, saturated alkyl divalentradical having the number of atoms indicated or when no atoms areindicated means a bond, e.g., (C₆-C₁₀)aryl(C₀-C₃)alkyl includes phenyl,benzyl, phenethyl, 1-phenylethyl 3-phenylpropyl, and the like. Backboneof the alkyl can be optionally inserted with one or more heteroatoms,such as N, O, or S. Examples of alkyl radicals include, but are notlimited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl,neopentyl, n-hexyl, and n-octyl radicals.

In preferred embodiments, a straight chain or branched chain alkyl has30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straightchains, C3-C30 for branched chains), and more preferably 20 or fewer.Likewise, preferred cycloalkyls have from 3-10 carbon atoms in theirring structure, and more preferably have 5, 6 or 7 carbons in the ringstructure. The term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having one or more substituents replacing ahydrogen on one or more carbons of the hydrocarbon backbone.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Throughout the application, preferred alkylgroups are lower alkyls. In preferred embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

Substituents of a substituted alkyl can include halogen, hydroxy, nitro,thiols, amino, azido, imino, amido, phosphoryl (including phosphonateand phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyland sulfonate), and silyl groups, as well as ethers, alkylthios,carbonyls (including ketones, aldehydes, carboxylates, and esters),—CF3,—CN and the like.

As used herein, the term “alkenyl” refers to unsaturated straight-chain,branched-chain or cyclic hydrocarbon radicals having at least onecarbon-carbon double bond. C_(x) alkenyl and C_(x)-C_(y)alkenyl aretypically used where X and Y indicate the number of carbon atoms in thechain. For example, C₂-C₆alkenyl includes alkenyls that have a chain ofbetween 1 and 6 carbons and at least one double bond, e.g., vinyl,allyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl,2-methylallyl, 1-hexenyl, 2-hexenyl, 3- hexenyl, and the like). Alkenylrepresented along with another radical (e.g., as in arylalkenyl) means astraight or branched, alkenyl divalent radical having the number ofatoms indicated. Backbone of the alkenyl can be optionally inserted withone or more heteroatoms, such as N, O, or S.

As used herein, the term “alkynyl” refers to unsaturated hydrocarbonradicals having at least one carbon-carbon triple bond. C_(x) alkynyland C_(x)-C_(y)alkynyl are typically used where X and Y indicate thenumber of carbon atoms in the chain. For example, C₂-C₆alkynyl includesalkynls that have a chain of between 1 and 6 carbons and at least onetriple bond, e.g., ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,isopentynyl, 1,3-hexa-diyn-yl, n-hexynyl, 3-pentynyl, 1-hexen-3-ynyl andthe like. Alkynyl represented along with another radical (e.g., as inarylalkynyl) means a straight or branched, alkynyl divalent radicalhaving the number of atoms indicated. Backbone of the alkynyl can beoptionally inserted with one or more heteroatoms, such as N, O, or S.

As used herein, the term “halogen” or “halo” refers to an atom selectedfrom fluorine, chlorine, bromine and iodine. The term “halogenradioisotope” or “halo isotope” refers to a radionuclide of an atomselected from fluorine, chlorine, bromine and iodine. A“halogen-substituted moiety” or “halo-substituted moiety”, as anisolated group or part of a larger group, means an aliphatic, alicyclic,or aromatic moiety, as described herein, substituted by one or more“halo” atoms, as such terms are defined in this application. Forexample, halo-substituted alkyl includes haloalkyl, dihaloalkyl,trihaloalkyl, perhaloalkyl and the like (e.g. halosubstituted(C₁-C₃)alkyl includes chloromethyl, dichloromethyl, difluoromethyl,trifluoromethyl (—CF₃), 2,2,2-trifluoroethyl, perfluoroethyl,2,2,2-trifluoro-1,1-dichloroethyl, and the like).

The term “cyclyl” or “cycloalkyl” refers to saturated and partiallyunsaturated cyclic hydrocarbon groups having 3 to 12 carbons, forexample, 3 to 8 carbons, and, for example, 3 to 6 carbons. C_(x)cyclyland C_(x)-C_(y)cylcyl are typically used where X and Y indicate thenumber of carbon atoms in the ring system. The cycloalkyl groupadditionally can be optionally substituted, e.g., with 1, 2, 3, or 4substituents. Examples of cyclyl groups include, without limitation,cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl,cyclohexenyl, 2,5-cyclohexadienyl, cycloheptyl, cyclooctyl,bicyclo[2.2.2]octyl, adamantan-1-yl, decahydronaphthyl, oxocyclohexyl,dioxocyclohexyl, thiocyclohexyl, 2-oxobicyclo [2.2.1]hept-1-yl, and thelike

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively). C_(x)heterocyclyl andC_(x)-C_(y)heterocyclyl are typically used where X and Y indicate thenumber of carbon atoms in the ring system. In some embodiments, 1, 2 or3 hydrogen atoms of each ring can be substituted by a substituent.Exemplary heterocyclyl groups include, but are not limited topiperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl,piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl,perhydropyrrolizinyl, 1,4-diazaperhydroepinyl, 1,3-dioxanyl,1,4-dioxanyland the like.

The terms “bicyclic” and “tricyclic” refers to fused, bridged, or joinedby a single bond polycyclic ring assemblies. As used herein, the term“fused ring” refers to a ring that is bonded to another ring to form acompound having a bicyclic structure when the ring atoms that are commonto both rings are directly bound to each other. Non-exclusive examplesof common fused rings include decalin, naphthalene, anthracene,phenanthrene, indole, furan, benzofuran, quinoline, and the like.Compounds having fused ring systems can be saturated, partiallysaturated, cyclyl, heterocyclyl, aromatics, heteroaromatics, and thelike.

The term “aryl” refers to monocyclic, bicyclic, or tricyclic fusedaromatic ring system. C_(x) aryl and C_(x)-C_(y)aryl are typically usedwhere X and Y indicate the number of carbon atoms in the ring system.Exemplary aryl groups include, but are not limited to, pyridinyl,pyrimidinyl, furanyl, thienyl, imidazolyl, thiazolyl, pyrazolyl,pyridazinyl, pyrazinyl, triazinyl, tetrazolyl, indolyl, benzyl, phenyl,naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl,phenyl, tetrahydronaphthyl, benzimidazolyl, benzofuranyl,benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl,benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl,carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl,2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl,furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl,indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl,isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl,isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl,morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl,phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl,phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl,piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl,pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl and xanthenyl, and the like. In someembodiments, 1, 2, 3, or 4 hydrogen atoms of each ring can besubstituted by a substituent.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered fused bicyclic, or 11-14 membered fused tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively. C_(x)heteroaryl and C_(x)-C_(y)heteroaryl are typically used where X and Yindicate the number of carbon atoms in the ring system. Heteroarylsinclude, but are not limited to, those derived from benzo[b]furan,benzo[b] thiophene, benzimidazole, imidazo[4,5-c]pyridine, quinazoline,thieno[2,3-c]pyridine, thieno[3,2-b]pyridine, thieno[2, 3-b]pyridine,indolizine, imidazo[1,2a]pyridine, quinoline, isoquinoline, phthalazine,quinoxaline, naphthyridine, quinolizine, indole, isoindole, indazole,indoline, benzoxazole, benzopyrazole, benzothiazole,imidazo[1,5-a]pyridine, pyrazolo[1,5-a]pyridine,imidazo[1,2-a]pyrimidine, imidazo[1,2-c]pyrimidine,imidazo[1,5-a]pyrimidine, imidazo[1,5-c]pyrimidine,pyrrolo[2,3-b]pyridine, pyrrolo[2,3cjpyridine, pyrrolo[3,2-c]pyridine,pyrrolo[3,2-b]pyridine, pyrrolo[2,3-d]pyrimidine,pyrrolo[3,2-d]pyrimidine, pyrrolo [2,3-b]pyrazine,pyrazolo[1,5-a]pyridine, pyrrolo[1,2-b]pyridazine,pyrrolo[1,2-c]pyrimidine, pyrrolo[1,2-a]pyrimidine,pyrrolo[1,2-a]pyrazine, triazo[1,5-a]pyridine, pteridine, purine,carbazole, acridine, phenazine, phenothiazene, phenoxazine,1,2-dihydropyrrolo[3,2,1-hi]indole, indolizine, pyrido[1,2-a]indole,2(1H)-pyridinone, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxepanyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl,phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl,phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl,4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl,pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. Someexemplary heteroaryl groups include, but are not limited to, pyridyl,furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl orthienyl, pyridazinyl, pyrazinyl, quinolinyl, indolyl, thiazolyl,naphthyridinyl, 2-amino-4-oxo-3,4-dihydropteridin-6-yl,tetrahydroisoquinolinyl, and the like. In some embodiments, 1, 2, 3, or4 hydrogen atoms of each ring may be substituted by a substituent.

As used herein, the term “substituted” refers to independent replacementof one or more of the hydrogen atoms on the substituted moiety withsubstituents independently selected from, but not limited to, alkyl,alkenyl, heterocycloalkyl, alkoxy, aryloxy, hydroxy, amino, amido,alkylamino, arylamino, cyano, halo, mercapto, nitro, carbonyl, acyl,aryl and heteroaryl groups.

As used herein, the term “substituted” refers to independent replacementof one or more (typically 1, 2, 3, 4, or 5) of the hydrogen atoms on thesubstituted moiety with substituents independently selected from thegroup of substituents listed below in the definition for “substituents”or otherwise specified. In general, a non-hydrogen substituent can beany substituent that can be bound to an atom of the given moiety that isspecified to be substituted. Examples of substituents include, but arenot limited to, acyl, acylamino, acyloxy, aldehyde, alicyclic,aliphatic, alkanesulfonamido, alkanesulfonyl, alkaryl, alkenyl, alkoxy,alkoxycarbonyl, alkyl, alkylamino, alkylcarbanoyl, alkylene, alkylidene,alkylthios, alkynyl, amide, amido, amino, amino, aminoalkyl, aralkyl,aralkylsulfonamido, arenesulfonamido, arenesulfonyl, aromatic, aryl,arylamino, arylcarbanoyl, aryloxy, azido, carbamoyl, carbonyl, carbonyls(including ketones, carboxy, carboxylates, CF₃, cyano (CN), cycloalkyl,cycloalkylene, ester, ether, haloalkyl, halogen, halogen, heteroaryl,heterocyclyl, hydroxy, hydroxy, hydroxyalkyl, imino, iminoketone,ketone, mercapto, nitro, oxaalkyl, oxo, oxoalkyl, phosphoryl (includingphosphonate and phosphinate), silyl groups, sulfonamido, sulfonyl(including sulfate, sulfamoyl and sulfonate), thiols, and ureidomoieties, each of which may optionally also be substituted orunsubstituted. In some cases, two substituents, together with thecarbon(s) to which they are attached to, can form a ring.

Aryl and heteroaryls can be optionally substituted with one or moresubstituents at one or more positions, for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl,carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF3, —CN, or thelike.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy, n-propyloxy, iso-propyloxy, n-butyloxy, iso-butyloxy, andthe like. An “ether” is two hydrocarbons covalently linked by an oxygen.Accordingly, the substituent of an alkyl that renders that alkyl anether is or resembles an alkoxyl, such as can be represented by one of-O-alkyl, -O-alkenyl, and -O-alkynyl. Aroxy can be represented by-O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as definedbelow. The alkoxy and aroxy groups can be substituted as described abovefor alkyl.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of -S-alkyl, -S-alkenyl, and-S-alkynyl. Representative alkylthio groups include methylthio,ethylthio, and the like. The term “alkylthio” also encompassescycloalkyl groups, alkene and cycloalkene groups, and alkyne groups.“Arylthio” refers to aryl or heteroaryl groups.

The term “sulfinyl” means the radical —SO—. It is noted that thesulfinyl radical can be further substituted with a variety ofsubstituents to form different sulfinyl groups including sulfinic acids,sulfinamides, sulfinyl esters, sulfoxides, and the like.

The term “sulfonyl” means the radical —SO₂—. It is noted that thesulfonyl radical can be further substituted with a variety ofsubstituents to form different sulfonyl groups including sulfonic acids(—SO₃H), sulfonamides, sulfonate esters, sulfones, and the like.

The term “thiocarbonyl” means the radical —C(S)—. It is noted that thethiocarbonyl radical can be further substituted with a variety ofsubstituents to form different thiocarbonyl groups including thioacids,thioamides, thioesters, thioketones, and the like.

As used herein, the term “amino” means —NH₂. The term “alkylamino” meansa nitrogen moiety having at least one straight or branched unsaturatedaliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen.For example, representative amino groups include —NH₂, —NHCH₃, —N(CH₃)₂,-NH(C₁-C₁₀alkyl), -N(C₁-C₁₀alkyl)₂, and the like. The term “alkylamino”includes “alkenylamino,” “alkynylamino,” “cyclylamino,” and“heterocyclylamino.” The term “arylamino” means a nitrogen moiety havingat least one aryl radical attached to the nitrogen. For example -NHaryl,and -N(aryl)₂. The term “heteroarylamino” means a nitrogen moiety havingat least one heteroaryl radical attached to the nitrogen. For example-NHheteroaryl, and -N(heteroaryl)₂. Optionally, two substituentstogether with the nitrogen can also form a ring. Unless indicatedotherwise, the compounds described herein containing amino moieties caninclude protected derivatives thereof. Suitable protecting groups foramino moieties include acetyl, tertbutoxycarbonyl, benzyloxycarbonyl,and the like.

The term “aminoalkyl” means an alkyl, alkenyl, and alkynyl as definedabove, except where one or more substituted or unsubstituted nitrogenatoms (—N—) are positioned between carbon atoms of the alkyl, alkenyl,or alkynyl . For example, an (C₂-C₆) aminoalkyl refers to a chaincomprising between 2 and 6 carbons and one or more nitrogen atomspositioned between the carbon atoms.

The term “alkoxyalkoxy” means -O-(alkyl)-O-(alkyl), such as—OCH₂CH₂OCH₃, and the like. The term “alkoxycarbonyl” means-C(O)O-(alkyl), such as —C(═O)OCH₃, —C(═O)OCH₂CH₃, and the like. Theterm “alkoxyalkyl” means -(alkyl)-O-(alkyl), such as —CH₂OCH₃,—CH₂OCH₂CH₃, and the like. The term “aryloxy” means -O-(aryl), such as-O-phenyl, -O-pyridinyl, and the like. The term “arylalkyl” means-(alkyl)-(aryl), such as benzyl (i.e., -CH₂phenyl), -CH₂-pyrindinyl, andthe like. The term “arylalkyloxy” means -O-(alkyl)-(aryl), such as-O-benzyl, -O-CH₂-pyridinyl, and the like. The term “cycloalkyloxy”means -O-(cycloalkyl), such as -O-cyclohexyl, and the like. The term“cycloalkylalkyloxy” means -O-(alkyl)-(cycloalkyl, such as-OCH₂cyclohexyl, and the like. The term “aminoalkoxy” means-O-(alkyl)-NH₂, such as —OCH₂NH₂, —OCH₂CH₂NH₂, and the like. The term“mono- or di-alkylamino” means -NH(alkyl) or -N(alkyl)(alkyl),respectively, such as —NHCH₃, —N(CH₃)₂, and the like. The term “mono- ordi-alkylaminoalkoxy” means -O-(alkyl)- NH(alkyl) or-O-(alkyl)-N(alkyl)(alkyl), respectively, such as —OCH₂NHCH₃,—OCH₂CH₂N(CH₃)₂, and the like. The term “arylamino” means -NH(aryl),such as -NH-phenyl, -NH-pyridinyl, and the like. The term“arylalkylamino” means -NH-(alkyl)-(aryl), such as -NH-benzyl,-NHCH₂-pyridinyl, and the like. The term “alkylamino” means -NH(alkyl),such as —NHCH₃, —NHCH₂CH₃, and the like. The term “cycloalkylamino”means -NH-(cycloalkyl), such as -NH-cyclohexyl, and the like. The term“cycloalkylalkylamino” -NH-(alkyl)-(cycloalkyl), such as-NHCH₂-cyclohexyl, and the like.

It is noted in regard to all of the definitions provided herein that thedefinitions should be interpreted as being open ended in the sense thatfurther substituents beyond those specified may be included. Hence, a C₁alkyl indicates that there is one carbon atom but does not indicate whatare the substituents on the carbon atom. Hence, a C₁ alkyl comprisesmethyl (i.e., —CH3) as well as -CR_(a)R_(b)R_(c) where R_(a), R_(b), andR_(c) caneach independently be hydrogen or any other substituent wherethe atom alpha to the carbon is a heteroatom or cyano. Hence, CF₃, CH₂OHand CH₂CN are all C₁ alkyls.

Unless otherwise stated, structures depicted herein are meant to includecompounds which differ only in the presence of one or more isotopicallyenriched atoms. For example, compounds having the present structureexcept for the replacement of a hydrogen atom by a deuterium or tritium,or the replacement of a carbon atom by a ¹³C- or ¹⁴C-enriched carbon arewithin the scope of the invention.

As used here in the term “isomer” refers to compounds having the samemolecular formula but differing in structure. Isomers which differ onlyin configuration and/or conformation are referred to as “stereoisomers.”The term “isomer” is also used to refer to an enantiomer.

The term “enantiomer” is used to describe one of a pair of molecularisomers which are mirror images of each other and non-superimposable.Other terms used to designate or refer to enantiomers include“stereoisomers” (because of the different arrangement or stereochemistryaround the chiral center; although all enantiomers are stereoisomers,not all stereoisomers are enantiomers) or “optical isomers” (because ofthe optical activity of pure enantiomers, which is the ability ofdifferent pure enantiomers to rotate planepolarized light in differentdirections). Enantiomers generally have identical physical properties,such as melting points and boiling points, and also have identicalspectroscopic properties. Enantiomers can differ from each other withrespect to their interaction with plane-polarized light and with respectto biological activity.

The term “racemic mixture”, “racemic compound” or “racemate” refers to amixture of the two enantiomers of one compound. An ideal racemic mixtureis one wherein there is a 50:50 mixture of both enantiomers of acompound such that the optical rotation of the (+) enantiomer cancelsout the optical rotation of the (-) enantiomer.

The term “resolving” or “resolution” when used in reference to a racemicmixture refers to the separation of a racemate into its twoenantiomorphic forms (i.e., (+) and (-); or (R) and (S) forms). Theterms can also refer to enantioselective conversion of one isomer of aracemate to a product.

The term “enantiomeric excess” or “ee” refers to a reaction productwherein one enantiomer is produced in excess of the other, and isdefined for a mixture of (+)- and (-)-enantiomers, with compositiongiven as the mole or weight or volume fraction F₍₊₎ and F₍₋₎ (where thesum of F₍₊₎ and F₍₋₎ = 1). The enantiomeric excess is defined as * F₍₊₎-F₍₋₎* and the percent enantiomeric excess by 100x* F₍₊₎ -F₍₋₎*. The“purity” of an enantiomer is described by its ee or percent ee value (%ee).

Whether expressed as a “purified enantiomer” or a “pure enantiomer” or a“resolved enantiomer” or “a compound in enantiomeric excess”, the termsare meant to indicate that the amount of one enantiomer exceeds theamount of the other. Thus, when referring to an enantiomer preparation,both (or either) of the percent of the major enantiomer (e.g. by mole orby weight or by volume) and (or) the percent enantiomeric excess of themajor enantiomer may be used to determine whether the preparationrepresents a purified enantiomer preparation.

The term “enantiomeric purity” or “enantiomer purity” of an isomerrefers to a qualitative or quantitative measure of the purifiedenantiomer; typically, the measurement is expressed on the basis of eeor enantiomeric excess.

The terms “substantially purified enantiomer”, “substantially resolvedenantiomer” “substantially purified enantiomer preparation” are meant toindicate a preparation (e.g. derived from non-optically active startingmaterial, substrate, or intermediate) wherein one enantiomer has beenenriched over the other, and more preferably, wherein the otherenantiomer represents less than 20%, more preferably less than 10%, andmore preferably less than 5%, and still more preferably, less than 2% ofthe enantiomer or enantiomer preparation.

The terms “purified enantiomer”, “resolved enantiomer” and “purifiedenantiomer preparation” are meant to indicate a preparation (e.g.derived from non-optically active starting material, substrates orintermediates) wherein one enantiomer (for example, the R-enantiomer) isenriched over the other, and more preferably, wherein the otherenantiomer (for example the S-enantiomer) represents less than 30%,preferably less than 20%, more preferably less than 10% (e.g. in thisparticular instance, the R-enantiomer is substantially free of theS-enantiomer), and more preferably less than 5% and still morepreferably, less than 2% of the preparation. A purified enantiomer maybe synthesized substantially free of the other enantiomer, or a purifiedenantiomer may be synthesized in a stereopreferred procedure, followedby separation steps, or a purified enantiomer may be derived from aracemic mixture.

The term “enantioselectivity”, also called the enantiomeric ratioindicated by the symbol “E”, refers to the selective capacity of anenzyme to generate from a racemic substrate one enantiomer relative tothe other in a product racemic mixture; in other words, it is a measureof the ability of the enzyme to distinguish between enantiomers. Anonselective reaction has an E of 1, while resolutions with E’s above 20are generally considered useful for synthesis or resolution. Theenantioselectivity resides in a difference in conversion rates betweenthe enantiomers in question. Reaction products are obtained that areenriched in one of the enantiomers; conversely, remaining substrates areenriched in the other enantiomer. For practical purposes it is generallydesirable for one of the enantiomers to be obtained in large excess.This is achieved by terminating the conversion process at a certaindegree of conversion.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” or “inhibit” typicallymeans a decrease by at least 10% as compared to a reference level (e.g.the absence of a given treatment or agent) and can include, for example,a decrease by at least about 10%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition orreduction as compared to a reference level. “Complete inhibition” is a100% inhibition as compared to a reference level. A decrease can bepreferably down to a level accepted as within the range of normal for anindividual without a given disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments, the terms “increased”, “increase”, “enhance”, or “activate”can mean an increase of at least 10% as compared to a reference level,for example an increase of at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level. In the context of amarker or symptom, a “increase” is a statistically significant increasein such level.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Insome embodiments, the subject is a mammal, e.g., a primate, e.g., ahuman. The terms, “individual,” “patient” and “subject” are usedinterchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models ofconditions described herein. A subject can be male or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatmentor one or more complications related to such a condition, andoptionally, have already undergone treatment for the condition or theone or more complications related to the condition. Alternatively, asubject can also be one who has not been previously diagnosed as havingthe condition or one or more complications related to the condition. Forexample, a subject can be one who exhibits one or more risk factors forthe condition or one or more complications related to the condition or asubject who does not exhibit risk factors.

A “subject in need” of treatment for a particular condition can be asubject having that condition, diagnosed as having that condition, or atrisk of developing that condition.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. “Protein” and“polypeptide” are often used in reference to relatively largepolypeptides, whereas the term “peptide” is often used in reference tosmall polypeptides, but usage of these terms in the art overlaps. Theterms “protein” and “polypeptide” are used interchangeably herein whenreferring to a gene product and fragments thereof. Thus, exemplarypolypeptides or proteins include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, fragments, and analogs of the foregoing.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The term also refers to antibodies comprised of twoimmunoglobulin heavy chains and two immunoglobulin light chains as wellas a variety of forms including full length antibodies andantigen-binding portions thereof; including, for example, animmunoglobulin molecule, a monoclonal antibody, a chimeric antibody, aCDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, aFv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), adiabody, a multispecific antibody, a dual specific antibody, ananti-idiotypic antibody, a bispecific antibody, a functionally activeepitope-binding portion thereof, and/or bifunctional hybrid antibodies.Each heavy chain is composed of a variable region of said heavy chain(abbreviated here as HCVR or VH) and a constant region of said heavychain. The heavy chain constant region consists of three domains CH1,CH2 and CH3. Each light chain is composed of a variable region of saidlight chain (abbreviated here as LCVR or VL) and a constant region ofsaid light chain. The light chain constant region consists of a CLdomain. The VH and VL regions may be further divided into hypervariableregions referred to as complementarity-determining regions (CDRs) andinterspersed with conserved regions referred to as framework regions(FR). Each VH and VL region thus consists of three CDRs and four FRswhich are arranged from the N terminus to the C terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure iswell known to those skilled in the art.

As used herein, the term “antibody reagent” refers to a polypeptide thatincludes at least one immunoglobulin variable domain or immunoglobulinvariable domain sequence and which specifically binds a given antigen.An antibody reagent can comprise an antibody or a polypeptide comprisingan antigen-binding domain of an antibody. In some embodiments, anantibody reagent can comprise a monoclonal antibody or a polypeptidecomprising an antigen-binding domain of a monoclonal antibody. Forexample, an antibody can include a heavy (H) chain variable region(abbreviated herein as VH), and a light (L) chain variable region(abbreviated herein as VL). In another example, an antibody includes twoheavy (H) chain variable regions and two light (L) chain variableregions. The term “antibody reagent” encompasses antigen-bindingfragments of antibodies (e.g., single chain antibodies, Fab and sFabfragments, F(ab′)2, Fd fragments, Fv fragments, scFv, and domainantibodies (dAb) fragments as well as complete antibodies.

Antibodies and/or antibody reagents can include an immunoglobulinmolecule, a monoclonal antibody, a chimeric antibody, a CDR-graftedantibody, a humanized antibody, a fully human antibody, a Fab, a Fab′, aF(ab′)2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody,a diabody, a multispecific antibody, a dual specific antibody, ananti-idiotypic antibody, a bispecific antibody, and a functionallyactive epitope-binding portion thereof.

As used herein, the term “nanobody” or single domain antibody (sdAb)refers to an antibody comprising the small single variable domain (VHH)of antibodies obtained from camelids and dromedaries. Antibody proteinsobtained from members of the camel and dromedary (Camelus baclrianus andCalelus dromaderius) family including new world members such as llamaspecies (Lama paccos, Lama glama and Lama vicugna) have beencharacterized with respect to size, structural complexity andantigenicity for human subjects. Certain IgG antibodies from this familyof mammals as found in nature lack light chains, and are thusstructurally distinct from the typical four chain quaternary structurehaving two heavy and two light chains, for antibodies from otheranimals. See PCT/EP93/ 02214 (WO 94/04678 published 3 Mar. 1994; whichis incorporated by reference herein in its entirety).

A region of the camelid antibody which is the small single variabledomain identified as VHH can be obtained by genetic engineering to yielda small protein having high afiinity for a target, resulting in a lowmolecular weight antibody-derived protein known as a “camelid nanobody”.See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see also Stijlemans, B.et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14:440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; andLauwereys, M. et al. 1998 EMBO J. 17: 3512-3520; each of which isincorporated by reference herein in its entirety. Engineered librariesof camelid antibodies and antibody fragments are commercially available,for example, from Ablynx, Ghent, Belgium. As with other antibodies ofnon-human origin, an amino acid sequence of a camelid antibody can bealtered recombinantly to obtain a sequence that more closely resembles ahuman sequence, i.e., the nanobody can be “humanized”. Thus the naturallow antigenicity of camelid antibodies to humans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth thatof a human IgG molecule and the protein has a physical diameter of onlya few nanometers. One consequence of the small size is the ability ofcamelid nanobodies to bind to antigenic sites that are functionallyinvisible to larger antibody proteins, i.e., camelid nanobodies areuseful as reagents detect antigens that are otherwise cryptic usingclassical immunological techniques, and as possible therapeutic agents.Thus yet another consequence of small size is that a camelid nanobodycan inhibit as a result of binding to a specific site in a groove ornarrow cleft of a target protein, and hence can serve in a capacity thatmore closely resembles the function of a classical low molecular weightdrug than that of a classical antibody. The low molecular weight andcompact size further result in camelid nanobodies being extremelythermostable, stable to extreme pH and to proteolytic digestion, andpoorly antigenic. See U.S. Pat. Application 20040161738 published Aug.19, 2004; which is incorporated by reference herein in its entirety.These features combined with the low antigenicity to humans indicategreat therapeutic potential.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid or an analog thereof.The nucleic acid can be either single-stranded or double-stranded. Asingle-stranded nucleic acid can be one nucleic acid strand of adenatured double- stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the nucleic acid can be DNA. In another aspect, thenucleic acid can be RNA. Suitable DNA can include, e.g., cDNA. SuitableRNA can include, e.g., mRNA.

As used herein, “inhibitory nucleic acid” refers to a nucleic acidmolecule which can inhibit the expression of a target, e.g.,double-stranded RNAs (dsRNAs), inhibitory RNAs (iRNAs), and the like.

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). The inhibitory nucleic acids described herein caninclude an RNA strand (the antisense strand) having a region which is 30nucleotides or less in length, i.e., 15-30 nucleotides in length,generally 19-24 nucleotides in length, which region is substantiallycomplementary to at least part the targeted mRNA transcript. The use ofthese iRNAs enables the targeted degradation of mRNA transcripts,resulting in decreased expression and/or activity of the target.

As used herein, the term “iRNA” refers to an agent that contains RNA (ormodified nucleic acids as described below herein) and which mediates thetargeted cleavage of an RNA transcript via an RNA-induced silencingcomplex (RISC) pathway. In some embodiments of any of the aspects, aniRNA as described herein effects inhibition of the expression and/oractivity of a target. In some embodiments of any of the aspects,contacting a cell with the inhibitor (e.g. an iRNA) results in adecrease in the target mRNA level in a cell by at least about 5%, about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, about 95%, about 99%, up to and including 100% ofthe target mRNA level found in the cell without the presence of theiRNA. In some embodiments of any of the aspects, administering aninhibitor (e.g. an iRNA) to a subject results in a decrease in thetarget mRNA level in the subject by at least about 5%, about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, about 95%, about 99%, up to and including 100% of the targetmRNA level found in the subject without the presence of the iRNA.

In some embodiments of any of the aspects, the iRNA can be a dsRNA. AdsRNA includes two RNA strands that are sufficiently complementary tohybridize to form a duplex structure under conditions in which the dsRNAwill be used. One strand of a dsRNA (the antisense strand) includes aregion of complementarity that is substantially complementary, andgenerally fully complementary, to a target sequence. The target sequencecan be derived from the sequence of an mRNA formed during the expressionof the target, e.g., it can span one or more intron boundaries. Theother strand (the sense strand) includes a region that is complementaryto the antisense strand, such that the two strands hybridize and form aduplex structure when combined under suitable conditions. Generally, theduplex structure is between 15 and 30 base pairs in length inclusive,more generally between 18 and 25 base pairs in length inclusive, yetmore generally between 19 and 24 base pairs in length inclusive, andmost generally between 19 and 21 base pairs in length, inclusive.Similarly, the region of complementarity to the target sequence isbetween 15 and 30 base pairs in length inclusive, more generally between18 and 25 base pairs in length inclusive, yet more generally between 19and 24 base pairs in length inclusive, and most generally between 19 and21 base pairs in length nucleotides in length, inclusive. In someembodiments of any of the aspects, the dsRNA is between 15 and 20nucleotides in length, inclusive, and in other embodiments, the dsRNA isbetween 25 and 30 nucleotides in length, inclusive. As the ordinarilyskilled person will recognize, the targeted region of an RNA targetedfor cleavage will most often be part of a larger RNA molecule, often anmRNA molecule. Where relevant, a “part” of an mRNA target is acontiguous sequence of an mRNA target of sufficient length to be asubstrate for RNAi-directed cleavage (i.e., cleavage through a RISCpathway). dsRNAs having duplexes as short as 9 base pairs can, undersome circumstances, mediate RNAi-directed RNA cleavage. Most often atarget will be at least 15 nucleotides in length, preferably 15-30nucleotides in length.

Exemplary embodiments of types of inhibitory nucleic acids can include,e.g,. siRNA, shRNA,miRNA, and/or amiRNA, which are well known in theart. In some embodiments of any of the aspects, the RNA of an iRNA,e.g., a dsRNA, is chemically modified to enhance stability or otherbeneficial characteristics. The nucleic acids described herein may besynthesized and/or modified by methods well established in the art, suchas those described in “Current protocols in nucleic acid chemistry,”Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY,USA, which is hereby incorporated herein by reference. Modificationsinclude, for example, (a) end modifications, e.g., 5′ end modifications(phosphorylation, conjugation, inverted linkages, etc.) 3′ endmodifications (conjugation, DNA nucleotides, inverted linkages, etc.),(b) base modifications, e.g., replacement with stabilizing bases,destabilizing bases, or bases that base pair with an expanded repertoireof partners, removal of bases (abasic nucleotides), or conjugated bases,(c) sugar modifications (e.g., at the 2′ position or 4′ position) orreplacement of the sugar, as well as (d) backbone modifications,including modification or replacement of the phosphodiester linkages.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder, e.g. a condition or disease described herein. The term“treating” includes reducing or alleviating at least one adverse effector symptom of a condition, disease or disorder. Treatment is generally“effective” if one or more symptoms or clinical markers are reduced.Alternatively, treatment is “effective” if the progression of a diseaseis reduced or halted. That is, “treatment” includes not just theimprovement of symptoms or markers, but also a cessation of, or at leastslowing of, progress or worsening of symptoms compared to what would beexpected in the absence of treatment. Beneficial or desired clinicalresults include, but are not limited to, alleviation of one or moresymptom(s), diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, remission (whetherpartial or total), and/or decreased mortality, whether detectable orundetectable. The term “treatment” of a disease also includes providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment).

As used herein, the term “pharmaceutical composition” refers to the atleast one IL as described herein in combination with a pharmaceuticallyacceptable carrier e.g. a carrier commonly used in the pharmaceuticalindustry. The phrase “pharmaceutically acceptable” is employed herein torefer to those compounds, materials, compositions, and/or dosage formswhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio. In someembodiments of any of the aspects, a pharmaceutically acceptable carriercan be a carrier other than water. In some embodiments of any of theaspects, a pharmaceutically acceptable carrier can be a cream, emulsion,gel, liposome, nanoparticle, and/or ointment. In some embodiments of anyof the aspects, a pharmaceutically acceptable carrier can be anartificial or engineered carrier, e.g., a carrier that the activeingredient would not be found to occur in in nature.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject.

As used herein, “contacting” refers to any suitable means fordelivering, or exposing, an agent to at least one cell. Exemplarydelivery methods include, but are not limited to, direct delivery tocell culture medium, perfusion, injection, or other delivery method wellknown to one skilled in the art. In some embodiments, contactingcomprises physical human activity, e.g., an injection; an act ofdispensing, mixing, and/or decanting; and/or manipulation of a deliverydevice or machine.

The term “effective amount” means an amount of a composition sufficientto provide at least some amelioration of the symptoms associated withthe condition. In one embodiment, the “effective amount” means an amountof a composition would decrease the markers or symptoms of the conditionin a subject having the condition.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean ±1%.

As used herein, the term “comprising” or “comprises” is used inreference to methods and compositions, and respective component(s)thereof, that are essential to the invention, yet open to the inclusionof unspecified elements, whether essential or not. As used herein, theterm “comprising” means that other elements can also be present inaddition to the defined elements presented. The use of “comprising”indicates inclusion rather than limitation.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

As used herein, the term “specific binding” refers to a chemicalinteraction between two molecules, compounds, cells and/or particleswherein the first entity binds to the second, target entity with greaterspecificity and affinity than it binds to a third entity which is anon-target. In some embodiments, specific binding can refer to anaffinity of the first entity for the second target entity which is atleast 10 times, at least 50 times, at least 100 times, at least 500times, at least 1000 times or greater than the affinity for the thirdnontarget entity. A reagent specific for a given target is one thatexhibits specific binding for that target under the conditions of theassay being utilized.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011(ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), TheEncyclopedia of Molecular Cell Biology and Molecular Medicine, publishedby Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8);Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway’sImmunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor& Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin’s GenesXI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: ALaboratory Manual, 4^(th) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., BasicMethods in Molecular Biology, Elsevier Science Publishing, Inc., NewYork, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology:DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); CurrentProtocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), JohnWiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocolsin Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons,Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan,ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe,(eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737),the contents of which are all incorporated by reference herein in theirentireties.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   1. A composition comprising at least one ionic liquid comprising a    quaternary ammonium cation and an undecanoic acid anion.-   2. The composition of paragraph 1, wherein the cation has a molar    mass equal to or greater than choline.-   3. The composition of any of the preceding paragraphs, wherein the    quaternary ammonium has the structure of NR₄ ⁺ and at least one R    group comprises a hydroxy group.-   4. The composition of any of the preceding paragraphs, wherein the    quaternary ammonium has the structure of NR₄ ⁺ and only one R group    comprises a hydroxy group.-   5. The composition of any of the preceding paragraphs, wherein the    cation is choline, C1, C6, or C7.-   6. The composition of any of the preceding paragraphs, wherein the    ionic liquid is at a concentration of at least 0.1%w/v.-   7. The composition of any of the preceding paragraphs, wherein the    ionic liquid is at a concentration of from about 10 to about 70%w/v.-   8. The composition of any of the preceding paragraphs, wherein the    ionic liquid is at a concentration of from about 30 to about 50%w/v.-   9. The composition of any of the preceding paragraphs, wherein the    ionic liquid is at a concentration of from about 30 to about 40%w/v.-   10. The composition of any of the preceding paragraphs, wherein the    ionic liquid comprises a ratio of cation to anion of from about 2:1    to about 1:10.-   11. The composition of any of the preceding paragraphs, wherein the    ionic liquid comprises a ratio of cation to anion of from about 1:1    to about 1:4.-   12. The composition of any of the preceding paragraphs, wherein the    ionic liquid comprises a ratio of cation to anion of about 1:1.-   13. The composition of any of the preceding paragraphs, wherein the    ionic liquid has a cation:anion ratio of 1:1.-   14. The composition of any of the preceding paragraphs, wherein the    composition does not comprise an active agent other than the at    least one ionic liquid.-   15. The composition of any of the preceding paragraphs, wherein the    composition further comprises an active compound other than the at    least one ionic liquid.-   16. The composition of any of the preceding paragraphs, wherein the    composition is formulated for administration transdermally, to a    mucus membrane, orally, ocularly, subcutaneously, intradermally,    parenterally, intratumorally, or intravenously.-   17. The composition of paragraph 16, wherein the composition is    formulated for ocular administration.-   18. The composition of any of the preceding paragraphs, further    comprising one or more additional antifungal agents.-   19. The composition of paragraph 18, wherein the one or more    additional antifungal agents are selected from the group consisting    of:    -   Amphotericin B; natamycin; voriconazole; povidone-iodine;        hypochlorous acid;    -   Chlorhexidine digluconate (CDG); vancomycin (VAN);        chloramphenicol (CHL);    -   polymyxin B (PMB); trimethoprim (TMP); benzalkonium chloride        (BAK); and combinations thereof.-   20. A method of treating or preventing a fungal infection,    comprising administering to a subject in need thereof a composition    of any of the preceding paragraphs.-   21. The method of any of the preceding paragraphs, wherein the    administration is transdermal, to a mucus membrane, oral,    subcutaneous, intradermal, parenteral, intratumoral, ocular, or    intravenous.-   22. The method of paragraph 21, wherein the fungal infection is an    ocular fungal infection and the composition is administered to one    or both eyes.-   23. The method of any of the preceding paragraphs, wherein the    subject is one who has received a corneal surgery.-   24. A method comprising:    -   a) performing corneal surgery on one or both eyes of a subject;        and    -   b) administering a composition of any of paragraphs 1-19 to the        one or both eyes.-   25. The method of any of the preceding paragraphs, wherein the    surgery is artificial cornea surgery.-   26. The method of any of the preceding paragraphs, wherein the    fungal infection is infectious keratitis and/or endopthalmitis.-   27. The method of any of the preceding paragraphs, wherein the    fungal infection is an infection of Candida, Candida albicans,    Candida parapsilosis, Fusarium, or Aspergillus.-   28. The method of any of the preceding paragraphs, wherein the    composition is administered daily.-   29. The method of any of the preceding paragraphs, wherein the    composition is provided in or on a contact lens, a lower    conjunctival fornix device, or a subconjunctival device.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

EXAMPLES Example 1

There is an unmet need for prophylaxis against fungal infectionsfollowing keratoprosthesis (KPro) implantation. Described herein is theevaluation of the antifungal properties of topical antibioticspreparation that are already being used successfully to preventbacterial endophthalmitis, as well as some promising antiseptics.

Several commonly used antibiotics were tested in vitro against Candidaalbicans (ATCC 24433), Fusarium solani (ATCC MYA-3636), and Aspergillusfumigatus (ATCC MYA-3626). Time kill activity and minimum inhibitoryconcentrations (MIC) were determined. Also the antiseptics benzalkoniumchloride (BAK), povidone-iodine (PI), and ionic liquids (ILs) consistingof a 1:1 mixture of choline and undecanoic acid, were tested. Toxicitywas assayed in vitro on cultures of human corneal epithelial cells,subjected to Trypan Blue staining. Adhesion and tissue invasionexperiments were also carried out, with or without γ-irradiation, and byanalysis with fluorescent microscopy.

Polymyxin B (PMB)/trimethoprim (TMP)/BAK (Polytrim®), PMB alone,gatifloxacin (GAT) with BAK (Zymaxid®), and same-concentration BAKalone, exhibited moderate antifungal activity in vitro. A log unitdilution markedly reduced efficacy. Moxifloxacin (MOX) or GAT withoutBAK, as well as TMP, vancomycin (VAN), and chloramphenicol (CHL), had noeffect. 1% PI had good efficacy/toxicity ratio and IL performed evenbetter. Polytrim® reduced adhesion of C. albicans to Kontur™ contactlenses, which are used for Gamma-irradiated corneas, without additionalagents, showed enhanced resistance to fungal invasion.

Of the antibiotic preparations already in use for bacterial prophylaxisafter KPro surgery, Polytrim® has broad spectrum antibacterial effectand is very inexpensive. Its fungicidal effect is caused by both PMB andBAK. Polytrim®’s effect on fungi is weak but may be sufficient forprophylaxis. PI, used for decades in ophthalmology, as a 1% solutionappears to be promising as a long-term antifungal. Hypochlorous acid haspreviously been shown to have promise. The here synthesizedcholine-undecanoate IL is effective and virtually non-toxic.

Introduction

One of the most serious and feared complications following artificialcornea surgery (KPro) is still infectious keratitis and/orendophthalmitis. The introduction of bandage soft contact lenses anddaily, low-dose prophylactic antibiotic drops postoperatively for lifehave sharply reduced the incidence of bacterial endophthalmitis, yetfungal infections remain as an issue to be addressed.¹⁻⁵ In the Northernhemisphere, the causative organisms have most commonly been Candidaalbicans and Candida parapsilosis. In the hot and humid climates ofunder-resourced countries, fungal infections pose a much moresignificant problem. There the causative organisms have most commonlybeen filamentous fungi such as Fusarium and Aspergillus.^(6,) ⁷ In arecent study on KPro use in India, it was noted that fungal etiology ofpostoperative endophthalmitis occurred in almost equal numbers as thosewith bacterial endophthalmitis.⁸

Thus, there is clearly an unmet need for an effective, inexpensive,simply applied antifungal prophylactic treatment. One way to fill thisneed would be to adopt existing, well-established antifungals withproven efficacy such as amphotericin B, natamycin, voriconazole, orothers. These medications would be expected to be very efficient asprophylaxis, but are challenged by the need for compounding, chemicalinstability with resulting need for frequent replacement, or high cost,in addition to adding to the burden of self-medication. Development ofresistance may also be a long-term problem.

Another possible route, already taken by several surgeons, might be touse an antiseptic eye drop like povidone-iodine (PI), which hasbroad-spectrum activity and has for decades been utilized in preparationfor ophthalmic surgery.⁹⁻¹² It has also been suggested for prophylacticantimicrobial use after KPro implantation,^(12,) ¹³ and (anecdotally)implemented by other KPro surgeons. However, ocular inflammation fromlong-term use has occurred. More recently, 0.01% hypochlorous acid(Avenova®; Novabay Pharmaceuticals; Emeryville, CA) has been evaluatedfor daily antifungal prophylaxis. It was found to have rapid fungicidaland sporicidal activity within one minute of exposure time, withbroad-spectrum activity against Candida, Aspergillus, and Fusarium invitro.² This agent is well tolerated and is FDA-approved for thetreatment of blepharitis. However, the manufacturing process is not asimple one, and thus, may introduce concerns of cost and instability.Chlorhexidine digluconate (CDG) and newer type of antimicrobial, ionicliquids (ILs), based on a combination of choline and a medium-chainfatty acid, undecanoic acid,¹⁴⁻¹⁹ are also promising candidateanti-fungals for potential prophylactic use.

A third possibility would be to examine antibiotic formulations alreadyin use for antibacterial prophylaxis and examine them for possibleantifungal activity, which would simplify the medication burden for thepatient and reduce cost. An indication that this route might be fruitfulcomes from a recent publication by Behlau et al, 2014 on the incidencerate of endophthalmitis after Boston KPro (B-KPro). In a cohort ofnon-autoimmune patients, treated with one drop of Polytrim® (polymyxin B(PMB) /trimethoprim (TMP) /benzalkonium chloride (BAK)) per day asantibacterial prophylaxis for four years, no infection (bacterial orfungal) was observed, whereas when other antibiotics (e.g.fluoroquinolones, FQ’s) were exclusively used, several fungal infectionsoccurred.²⁰ (One-tailed Fisher exact test: p=0.057; two-tailed: p=0.101;H. Lee, 2018)

For the B-KPro, the leading choices worldwide in the past forprophylactic antibiotic drops against bacteria have been Polytrim®,FQ’s, vancomycin (VAN), and chloramphenicol (CHL), with or without theaddition of BAK. These drugs as well as the above-mentioned antisepticcompounds are tested herein for efficacy and toxicity in vitro againstC. albicans, F. solani, and A. fumigatus, the most common causativeagents of KPro-related fungal infections.³

Materials and Methods Fungal Culture.

American Type Culture Collection (ATCC) strains of C. albicans (ATCC24433), F. solani (ATCC MYA3636) and A. fumigatus (ATCC MYA3626) werepurchased and used for testing. Yeasts were aerobically grown onSabouraud dextrose agar (Difco; Detroit, MI) at 37° C. for 24 h.Filamentous fungi were aerobically grown on potato dextrose agar (BectonVickinson; Cockeysville, MD) at 25 - 35° C. for 5 days.

Antimicrobial Agents

Polytrim® (Allergan; Irvine, CA) eye drops, PI solution (Ricca Chemical;Arlington, TX), and CDG (Sigma Aldrich; St Louis, MO) were purchased.PMB, TMP, BAK, GAT, MOX, VAN, and CHL (Sigma-Aldrich; St Louis, MO) wereprepared at the concentration of their commercially marketed eye droppreparations, and tested for quality control of antibacterial activityaccording to the Clinical Laboratory Standards Institutes (CLSI) methods(M100).²¹ Choline-based ionic liquids containing undecanoate in a 1:1ratio, were synthesized as described below, and a 100 mM stock solutionin Milli Q water was prepared. Test solutions were diluted in RoswellPark Memorial Institute-1640 broth (RPMI; buffered with3-(N-Morpholino)propanesulfonic acid, or MOPS, at pH 7.0; Mediatech;Manassas, VA) for the time kill experiments. Tonicities of the bestsolutions were determined.

In Vitro Time Kill Tests. Microtiter time kill tests were carried out invitro according to the published CLSI methods (M27-A2).²²

i) Yeast: The antibiotic solutions, at either full or ⅒ strength, andantiseptics were inoculated with 1x10⁵ - 2x10⁶ CFU/mL of C. albicans inRPMI broth (buffered with MOPS at pH 7.0; Mediatech; Manassas, VA) for0 - 15 min. The inoculum was prepared by adjustment of the opticaldensity at 600 nm (OD₆₀₀) to 1.0 (corresponding to approximately 1x10⁶CFU/mL), and dilution by 1:10 in the final solution. To confirm thestarting inoculum size, yeasts were plated on? and colonies countedafter growth for 24 hrs at 37° C. The test solutions were neutralized ateach time point by ten-fold dilution in Dey-Engley neutralizing broth(Sigma-Aldrich; St Louis, MO), and serially track diluted (PMID:9343684) on Sabouraud dextrose agar (six serial 1:10 dilutions, 0.01 mLeach) in technical triplicate. Colonies were counted and recordedfollowing aerobic incubation for 24 hrs at 37° C.

ii) Filamentous fungi: The antibiotic solutions, at either full or ⅒strength, and antiseptics were inoculated with approximately 2x10⁵ -2x10⁶ conidia/mL of F. solani or A. fumigatus in RPMI broth (bufferedwith MOPS at pH 7.0; Mediatech; Manassas, VA) for 0 - 15 min. Conidiawere collected by flooding the agar plates with 10 mL of sterile 1X PBSand 0.1% Tween® 20 (Sigma Aldrich; St Louis, MO). The solution wastransferred to a sterile conical tube, and particles were allowed tosettle for 10 min. The resulting supernatant was transferred to a newsterile conical tube and centrifuged at 3,000 x g for 5 min. Thesupernatant was discarded, the remaining pellet was re-suspended in RPMIbroth (buffered with MOPS at pH 7.0), and the OD₆₀₀ was adjusted to 1.0.In the final test solutions, at each time point the inoculum was diluted1:10 and neutralized in Dey-Engley neutralizing broth (Sigma-Aldrich; StLouis, MO). Neutralized cultures were then serially diluted and platedon Sabouraud dextrose agar (two serial 1:10 dilutions, 0.1 mL each) intechnical duplicate. Viable conidia were counted and recorded followingaerobic incubation for 48 hrs (or as soon as growth was visible) at 25-35° C.

Choline undecanoate (1:1). Choline bicarbonate (80% in water, SigmaAldrich) was combined, with vigorous stirring, with undecanoic acid(98%, Sigma Aldrich) in a 1:1 molar ratio at 40° C. The mixture was leftstirring overnight, then dried in a rotary evaporator at 10 mbar and 60°C. for 2 hrs, before being placed in a vacuum oven at 60° C. for 72 hrs.The resulting product was a malleable amber semisolid, and the chemicalidentity was confirmed by Nuclear Magnetic Resonance Spectroscopy, withpeaks identified as follows: ¹H NMR (600 MHz, d-DMSO) 0.84 (dt, 3H,OOCCH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₃); 1.22-1.36 (m, 16H,OOCCH₂CH₂(CH₂)₈CH₃); 1.72 (h, 2H, OOCCH₂CH₂(CH₂)₈CH₃); 3.31 (q, 2H,OOCCH₂CH₂(CH₂)₈CH₃); 3.08 (s, 9H, NCH₃); 3.37 (h, 2H, NCH₂CH₂OH); 3.82(h, 2H, NCH₂CH₂OH).

In Vitro Toxicity Tests. Trypan Blue assays for toxicity of variousantimicrobial agents were conducted using human corneal cells.Telomerase-immortalized human corneal-limbal epithelial cells (HCLE)were thawed at 37° C. and quickly transferred to a sterile T75 cellculture flask. Cells were grown in keratinocyte serum-free medium (KSFM;Gibco™; Thermofisher Scientific; Waltham, MA) supplemented with bovinepituitary extract (25 µg/mL), epidermal growth factor (0.2 ng/mL),calcium chloride (0.4 mM CaCl₂□2H₂O; Sigma-Aldrich; St Louis, MO), and1X penicillin/streptomycin, at 37° C. with 5% CO₂. Once cells wereconfluent, they were passaged following trypsinization in 12- or 24-wellplates. After incubation with Trypsin-EDTA (0.05%, Gibco™; ThermofisherScientific; Waltham, MA) for 5 - 10 min at 37° C. with 5% CO₂, thereaction was stopped with an equivalent volume of neutralizing medium(Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12, DMEM/F-12, withHEPES and 10% newborn calf serum), centrifuged at 5,000 x g for 5 min,and re-suspended in supplemented KSFM. Experiments were performed usingcells from passages 31 to 36. Once cells reached confluency, they weregently washed twice with DMEM/F-12 (Gibco™; Thermofisher Scientific;Waltham, MA) and incubated with an antimicrobial agent for 1 min (n=2wells per group). Controls included 1X PBS (diluent) and 1% TX-100 (cellpermeabilizer). After exposure to various agents, the cells were washedthree times with DMEM/F-12, incubated with 0.2% Trypan Blue for 5 min,and washed again three times with DMEM/F-12. The remaining media wasaspirated and replaced with 1X PBS for imaging. Macro-images of theculture plate were taken with a smartphone camera (iPhone 7; Apple Inc.;Cupertino, CA), and 10X images were taken with an inverted microscope(Eclipse TS100; Nikon; Tokyo, Japan) attached to a digital camera (SPOTInsight Fire Wire; Diagnostic Instruments Inc.; Sterling Heights, MI).3-6 images/well were obtained in clockwise direction from areasimmediately adjacent to the center of the well, and without overlappingareas. Images were quantified in ImageJ Fiji software to determine theamount of cell uptake of Trypan Blue. Macros were created from the ColorThreshoulder plugin (G. Landini) to batch process the images after colormatching in Photoshop to reference images per treatment group. % Area ofTrypan Blue staining per image was calculated as follows: (area ofblue/area of all) x 100%.

In Vitro Adhesion Tests. Polytrim® was diluted ⅒ and 1/100 in sterilePBS, and combined with C. albicans (OD₆₀₀ adjusted to 1.5) for 90 min at37° C. Following incubation, the cells were washed three times(centrifugation at 3000 x g for 5 min) and resuspended in PBS to removetrace amounts of the antibiotics. Kontur™ soft contact lenses (KonturKontact Lens; Hercules, CA) were placed in a sterile 24-well tissueculture plate (Corning; Corning, NY) containing 1 mL of the pre-treatedfungal cells. (Positive controls contained non-treated fungal cells, andnegative controls contained only sterile PBS.) The plates wereaerobically incubated for 24 hrs at 37° C. with gentle movement(machine, speed), and carefully washed three times with 1 mL PBS. Toremove fungal cells potentially adhering to the soft contact lenses, thetissue culture plate was sonicated in a sonifier bath (Branson2400;Branson Ultrasonics; Danbury, CT) for 1 min at maximum strength, andmanually agitated by nudging with a sterile micropipettor tip for anadditional 10 sec. The remaining solution was serially diluted, platedonto Sabouraud dextrose agar (0.1 mL) in technical triplicate, andviable colonies were counted and recorded following aerobic incubationfor 24 hrs at 37° C.

Ex Vivo Tissue Invasion Tests. Porcine globes were purchased(VisionTech; Mesquite, TX), and grossly examined to exclude eyes withdetectable corneal surface defects or stromal opacities. Corneoscleralrims were dissected using a surgical blade and fine scissors, gentlydenuded with a surgical blade and Weck-Cel® sponge (XOMED SurgicalProducts; Jacksonville, FL), and immersed in corneal storage medium withDMEM/F-12 (Gibco™; Thermofisher Scientific; Waltham, MA) and 5% dextran(500 kDa; Thermo Fisher Scientific; Waltham, MA). All eyes were storedat -80° C. before experiments. For treatment groups, the corneoscleralrims were gamma-irradiated at 25 kGy according to standard sterilizationprocedure. The treated and non-treated corneoscleral rims were immersedtogether in a solution with C. albicans (corneal storage medium with anOD₆₀₀ of 2.0), and incubated for 48 hrs at 37° C. with shaking (speed)on a (model). Smaller-size corneal buttons were obtained by dissectionwith a trephine (7 mm; Acuderm Inc.; Ft. Lauderdale, FL), andsubsequently washed in 1 mL of sterile 1X PBS. The buttons were embeddedin Tissue-TEK OCT compound (Sakura Finetek; Zoeterwoude, TheNetherlands), flash frozen on dry ice, and cut into 10 uM thick sections(machine). The sections were fixed in 4% paraformaldehyde, stained withcalcofluor white (Sigma-Aldrich; St Louis, MO), and examined under anepifluorescent microscope (Leica DM5500B; Leica Microsystems; BuffaloGrove, IL). Images (4X) were captured with a (camera type), and saved.

Results

To investigate the fungicidal activity of various antibiotics andantimicrobials, the reduction in fungal load after short contact times(0 - 15 min, FIGS. 2 - 3 ), to account for the effect of rapid dilutionby the tear film, as occurs in patients after topical instillation, wasmeasured. ²³ The detection limit for time kill tests was approximately100 CFU/mL for C. albicans, and 1 CFU/mL for F. solani and A. fumigatus(corresponding to a ≥3 log difference from the starting inoculum). 20 -300 cells were considered a reliable counting range per dilution, andvalues were averaged when more than one dilution in the same plate werewithin this range.

Polytrim® exhibits some fungicidal activity, attributable to both PMBand BAK.

Overall, depending on the particular testing conditions in vitro,Polytrim® was slowly fungicidal, and the activity could be attributed toboth PMB and BAK. Fungicidal activity was species-, contact time-, andconcentration dependent (FIG. 1A). For C. albicans, longer times (60-120 min) were required to cause a ≥1 - 2 log reduction in viable cells(data not shown). A small amount of activity was detectable after briefcontact (≤15 min; <10-fold reduction). PMB and BAK individually, werecomparable to the commercially prepared Polytrim®, whereas TMP as a soleagent had no effect within 120 min. When diluted 10-fold, neitherPolytrim® nor its components caused detectable killing within 120 min(FIG. 1B). Full strength Polytrim® was most effective against F. solani(FIG. 2B), causing a ≥3 log reduction (to below the detection limit) inviable conidia within 1 min, while A. fumigatus was the most resistant,requiring at least 15 - 120 min to achieve 1 - 3 logs of reduction.

Other commonly used topical antibiotics (0.5% MOX, 0.5% GAT, 0.5% CHL,and 1.4% VAN) prepared without BAK, had no effect against C. albicanswithin 120 min (FIG. 1C). Commercially prepared GAT (Zymaxid®; BAK0.005%, Allergan; Irvine, CA) was as effective as BAK alone in time killtests with. These results were consistent with findings from previousstudies that compared in vitro antifungal activity of antibioticsprepared with and without BAK.^(24,) ²⁵

PI exhibits rapid and potent fungicidal activity.

PI at concentrations of 0.5 - 1% exhibited rapid and potent fungicidalactivity against all of the tested fungal organisms in vitro (FIGS.2A-2D), consistent with its well-known broad-spectrum action.^(10,)^(11,) ²⁶ At higher concentrations (0.5 - 1%), PI required less time tokill C. albicans compared to the lower (0.01 - 0.25%), causing a ≥3 logreduction within 1 min, while 0.01 - 0.1% did not exhibit notableactivity (<1 log) within 120 min. These results indicate that povidoneiodine, in the range of 0.5 - 1%, can provide rapid and broad killing ofboth bacteria and fungi.

Choline-undecanoate exhibits rapid and potent fungicidal activity, andlow toxicity.

Choline-undecanoate exhibited in vitro fungicidal activity within 1 - 15min that was species-, contact time-, and concentration-dependent.Contact with 10 mM of this IL caused a ≥ 1 - 4 log reduction in viableC. albicans, F. solani, and A. fumigatus within 1 min (FIGS. 2A-2D).

Cytotoxicity and efficacy/toxicity ratio.

The calculated ratios of fungicidal activity and cytotoxicity, shown inFIG. 3 , represent their effect against fungi in relation to HCLE cellviability following 1 min of contact. Thus, a higher ratio is indicativeof higher safety, and suggests that any immediate damage from topicalinstillation would be greater against fungal cells than that of hostepithelial cells. Theoretically, the cationic component initiatesinteraction with the cell membrane, allowing for subsequent insertionand/or disruption by the anionic component.¹⁹ FIG. 4 shows the chemicalstructure of the choline-undecanoate IL used in this study.

Choline-undecanoate IL (10 mM) showed greatest differential killing(ratios >1) for all three fungal species tested, listed in increasingorder: A. fumigatus, F solani, and C. albicans. In contrast, PI andPolytrim® do not show the same ordering and rank highest for F. solani.The fungicidal activity of PI was less variable between the testedspecies, and ranks second after the novel IL. Polytrim® has a profilethat is more species-dependent, ranking the lowest for C. albicans, butcomparable to that of PI and the novel IL for F. solani.

Well-known antiseptic agents, such as CDG and myristamidopropyldimethylamine (MAPD or Aldox), were also included in the analysis, andfound to rank lower than PI. Aldox is an amidoamine antiseptic withreported anti-acanthamoeba activity.²⁷ There are studies comparing itsactivity to polyhexamethylene biguanide (PHMB) or polyquaternium-1(PQ-1), which are variations of main ingredients contained in commercialcontact lens solutions.^(28,) ²⁹ In this study, CDG and Aldox exhibitedrapid and broad fungicidal activity, but their low ratios derive fromtheir high cytotoxicity to HCLE cells as was measured by post-exposureTrypan blue staining. As such, a low ratio does not necessarily indicatea lack of efficacy, but could also represent a broadly damaging effectfor both the host and pathogen cells.

Polytrim®-treated C. albicans and Adhesion to Soft Contact Lenses

Pre-treated C. albicans exhibited reduced adherence to the surface ofKontur™ soft contact lenses after overnight incubation at 37° C.Compared to untreated C. albicans controls, there was a 77% (⅒ strengthPolytrim® pretreatment) and 64% (1/100 strength pretreatment) reductionin adherent fungal cells (FIG. 5 ). Although ⅒ dilution of Polytrim® didnot exhibit fungicidal activity for C. albicans in vitro (FIG. 1B),contact with C. albicans for 90 min resulted in less fungal celladhesion on soft contact lenses recommended for extended wear in B-KPropatients following surgery. This difference was also present at thelowest tested concentration (1/100 strength), showing thatsub-fingicidal amounts of Polytrim® can prevent fungal adhesion.

Fungal Penetration of Gamma-irradiated Pig Cornea

Experiments with gamma-irradiated pig corneas showed that they wereintrinsically more resistant to penetration by C. albicans (ATCC 24433)than to non-treated pig corneas (FIG. 6 ). Hyphal infiltration wasobserved as a faint blue pattern (calcofluor white) within the denudedstromal tissue layers. This staining pattern was observed inapproximately a half-depth layer of non-treated corneas after 48 hrs ofaerobic incubation at 37° C., while it was absent in thegamma-irradiated corneas. Some stiffness of the tissue was also noted inhandling the treated corneas, which is consistent with previous findingsfrom another study of gamma-irradiated pig eyes.³⁰

Discussion

Surgical replacement of the cornea with a prosthesis is a proven methodfor restoring vision in eyes for which other therapies either fail orare not possible. Particularly in limited resource countries in warm andhumid climates, practical, effective, low toxicity, and inexpensiveantifungal prophylaxis regimen after KPro implantation would be ofconsiderable value in extending the utility of this surgicalintervention.

Described herein is the evaluation of the possible antifungal effect ofantimicrobials that are already in use to prevent bacterialendophthalmitis, including antibiotics and promising antiseptics.Antifungals, which would be too expensive or otherwise impractical touse prophylactically in KPro eyes over a lifetime around the world werenot examined.

Based on unfavorable efficacy/toxicity ratio, or cost or stabilityissues, some of the tested substances can be eliminated outright. Thus,the antibiotics FQ’s, VAN, CHL, and TMP, often very effective againstbacteria, were found to be ineffective against the fungi tested. Theantiseptic CDG, although effective, was too toxic in tests conductedhere to be likely practical for years-long prophylaxis.

Remaining candidates include:

-   1) PMB + BAK in the form of Polytrim®, or equivalent, although when    tested in vitro, the effect was weak. However, it has been used for    years in large numbers of B-KPro patients with good tolerance. There    are hints of antifungal effect sufficient for prophylaxis, even in    low doses, and a large-scale field trial in developing countries may    be warranted. Thus, it is possible that Polytrim® (or equivalent),    one drop once or twice a day, with high compliance, can effectively    prevent both bacterial and fungal infections.-   2) Of the antiseptics, PI, in concentrations of 0.5 - 5% has been    used safely for various purposes in ophthalmology for decades.²⁶ A    5% solution is acutely painful when instilled into the eye, but a 1%    concentration is well tolerated. It does not select microbes for    resistance,³¹ and it has some effect on biofilms.^(32,33) To our    knowledge, there are only a few reports using PI on a chronic basis    after KPro implantation or in blepharitis.^(12,) ³⁴ However, a    prophylactic regime of 1% PI, one drop daily or weekly, could prove    useful, with the caveat that prophylaxis be stopped if inflammation    should set in. This medication would add a second medication to the    patient’s regimen (not so for Polytrim®, which would could have a    “two-in-one” benefit).-   3) Hypochlorous acid (0.01% Avenova®) has been previously tested    extensively in our unit and been found to be highly effective    against fungi, and easily tolerated.² It is FDA-approved for    blepharitis and could be an alternative. One drop daily,    prophylactically, could be adequate. However, current high cost and    some chemical instability could pose limitations.-   4) The novel ILs show considerable promise in that their    efficacy/toxicity ratio can be very favorable.¹⁷ The    choline-undecanoate possesses wide utility as an antimicrobial. This    new class of antimicrobial can be a valuable agent for long term,    low toxicity, low cost K-pro prophylaxis.

In spite of the weak antifungal effect by Polytrim® and similar drugs invitro, they are well tested and may provide prophylactic effect as afirst line agent. Should the growing experience indicated that greaterprotection against fungal infection is needed, 1% PI, although somewhatuntested for chronic use, could be of value as a secondary line oftreatment (a drop a day, or up to 7 days). The availability of thesecurrently approved and inexpensive agents should give other promisingdrugs time for further development and testing.

Finally, of interest are the observations that treatment with Polytrim®appears to reduce fungal adhesion to soft contact lenses, and thatgamma-irradiated pig corneas exhibit substantially more resistance tofungal penetration than non-treated corneas. The latter may be ofconsiderable value since the use of gamma-irradiated carrier tissue forthe B-KPro will increase markedly with introduction of pre-assembledKPro-graft combinations, followed by radiation, resulting in a morepractical, safer, and more easily stored and shipped product.³⁵

A major problem with any microbial prophylaxis in KPro patients has beenthe need for long-term uninterrupted compliance with the medication tomake the outcome safe. The patient must instill a drop into the eye atleast once daily-for life-a task that can be difficult to adhere to,especially in resource-poor countries where the vast majority of thecorneal blind patients live.

In the longer term, the problem of prophylaxis compliance may be reducedby use of a drug-eluting contact lens (Ciolino), or a device withsimilar long-term effect that can reliably be placed in the lowerconjunctival fornix, or implanted subconjunctivally.³⁶

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What is claimed herein is:
 1. A composition comprising at least oneionic liquid comprising a quaternary ammonium cation and a undecanoicacid anion.
 2. The composition of claim 1, wherein the cation has amolar mass equal to or greater than choline.
 3. The composition of claim1, wherein the quaternary ammonium has the structure of NR₄ ⁺ and atleast one R group comprises a hydroxy group.
 4. The composition of claim1, wherein the cation is choline, C1, C6, or C7.
 5. The composition ofclaim 1, wherein the ionic liquid comprises a ratio of cation to anionof from 2:1 to 1:10.
 6. The composition of claim 1, wherein the ionicliquid comprises a ratio of cation to anion of from 2:1 to 1:2.
 7. Thecomposition of claim 1, wherein the ionic liquid has a cation:anionratio of 1:1.
 8. The composition of claim 1, wherein the compositiondoes not comprise an active agent other than the at least one ionicliquid.
 9. The composition of claim 1, wherein the composition isformulated for ocular administration.
 10. The composition of claim 1,further comprising one or more additional antifungal agents.
 11. Thecomposition of claim 10, wherein the one or more additional antifungalagents are selected from the group consisting of: Amphotericin B;natamycin; voriconazole; povidone-iodine; hypochlorous acid;Chlorhexidine digluconate (CDG); vancomycin (VAN); chloramphenicol(CHL); polymyxin B (PMB); trimethoprim (TMP); benzalkonium chloride(BAK); and combinations thereof. 12-20. (canceled)
 21. The compositionof claim 1, wherein the composition comprises a therapeuticallyeffective amount of the at least one ionic liquid to treat a fungalinfection in a subject in need thereof.
 22. The composition of claim 21,wherein the fungal infection is an ocular fungal infection.
 23. Thecomposition of claim 21, wherein the subject has received a cornealsurgery.
 24. The composition of claim 23, wherein the surgery isartificial cornea surgery.
 25. The composition of claim 21, wherein thefungal infection is infectious keratitis and/or endopthalmitis.
 26. Thecomposition of claim 21, wherein the fungal infection is an infection ofCandida, Candida albicans, Candida parapsilosis, Fusarium, orAspergillus.
 27. The composition of claim 21, wherein the composition iseffective to treat the fungal infection in the subject by dailyadministration to the subject.
 28. The composition of claim 21, whereinthe composition is formulated in or on a contact lens, a lowerconjunctival fornix device, or a subconjunctival device.